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Engineering specificity and activity of thermolysin-like proteases from Bacillusde Kreij, Arno

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I

Engineering specificity and activityof thermolysin-like proteases from

Bacillus

Arno de Kreij

II

Cover art.The front cover shows the active site cleft of thermolysin with a modeled substrate, based onseveral crystal structures of different inhibitors bound to thermolysin. The Zn2+ binding ligands andthe Zn2+ ion are shown in red. The catalytically active residues are shown in green. The S1' residuesare shown in yellow and the blue spheres represent Ca2+ ions. This picture is a more detailed viewof Figure 4.1 on page 43. Ray tracing of this image was performed by M.L. van Roosmalen.

The back cover shows a ribbon diagram of thermolysin. The red arrows represent β-sheets, whereasthe blue helices represent α-helices. The centre shows the central α-helix which is the bottom of theactive site cleft with the catalytic Zn2+ ion (large sphere). Four Ca2+ ions (small spheres), involvedin stability, are also shown. This picture is a colour representation of Figure 2.2 on page 26.

Druk: PrinPartners Ipskamp, Enschede

III

RIJKSUNIVERSITEIT GRONINGEN

Engineering specificity and activityof thermolysin-like proteases from

Bacillus

Proefschrift

ter verkrijging van het doctoraat in deWiskunde en Natuurwetenschappenaan de Rijksuniversiteit Groningen

op gezag van deRector Magnificus, dr. D.F.J. Bosscher,

in het openbaar te verdedigen opvrijdag 6 juli 2001

om 16.00 uur

door

Arno de Kreij

geboren op 27 maart 1970te Capelle aan den IJssel

IV

Promotores: Prof. dr. G. Venema.Prof. dr. ir. V.G.H. Eijsink.

Beoordelingscommissie: Prof. dr. O.P. Kuipers.Prof. dr. G. Vriend.Prof. dr. B.W. Dijkstra.

1

ContentsChapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

Chapter 9

Introduction

Substrate specificity in the highly heterogeneous M4 peptidasefamily is determined by a small subset of amino acids.

Probing Catalytic Hinge-bending Motions In Thermolysin-LikeProteases By Gly ����Ala Mutations.

The effect of changing the hydrophobic S1' subsite ofthermolysin-like proteases on substrate specificity.

Engineering the catalysis of a thermolysin-like protease bymodification of its surface charge.

Summary and general discussion.

Samenvatting en algemene discussie.

Literature references

List of publications

Nawoord

3

21

31

41

51

61

65

69

75

77

Introduction

3

Introduction

1. Introduction.1.1 General.

Enzymes offer many advantages overtraditional chemical catalysts. They are clean,often cheap, and renewable. However, fullexploitation of enzymes in industry requires themodification of enzymes in such a way that theyperform a desired reaction under a set ofspecific conditions. The problems that have tobe solved to be able to rationally modifyenzymes are diverse and relate to questions suchas: why are proteins folded the way they are,which structural features determine proteinstability, how does enzymatic catalysis work,and which structural elements control thespecificity of an enzyme? Protein engineering isthe multi-disciplinary approach to answer thesequestions by studying the structure-functionrelationships of proteins. Once these structure-function relationships are known, the obtainedknowledge can be exploited to endow proteinswith changed properties. Since the breakthroughof recombinant DNA technology tremendousprogress has been made in solving structure-function relationships. For example, theunderstanding of thermostability has led toalgorithms that can predict which changes in theprimary structure are likely to improve thethermostability of a protein (1). Proteinengineering could eventually enable the de novodesign of virtually any enzymatic property.

However, fully de novo design of anenzyme is still far away, and many questions onthe structure-function relationships of enzymesremain unanswered. One of the most importantsteps of the last decade in exploiting enzymes isundoubtedly the development of molecular

breeding strategies (2), in which gene splicingand random mutagenesis are combined to yieldenzymes with novel or strongly improvedproperties. Molecular breeding and otherrandom mutagenesis methods (3) are greatlyfacilitated by the advent of robotics, enablingthe handling and screening of tens of thousandsof mutants.

However impressive the results withrandom mutagenesis methods are in yieldingnovel or improved biocatalysts, they do notdirectly contribute to the knowledge of howproteins work. Reverse engineering, that isobtaining an enzyme with a novel functionalitythrough random mutagenesis and reverting itstep by step to the wild-type lacking thisfunctionality, would help in revealingcomplicated structure function-relationshipswhile at the same time yielding many newbiocatalysts.

This thesis describes the developmentand use of protein engineering technologies tochange the properties of thermolysin-likeproteases produced by various Bacillus species.

1.2 The exploitation of the genus Bacilluswith respect to industrially importantenzymes.

The Gram-positive family ofBacillaceae contains four aerobic, endospore-forming genera i.e. Thermoactinomyces,Sporosarcina, Sporolactobacillus and Bacillus(4). Compared to the three other genera, thegenus Bacillus is quite heterogeneous: itconsists of all the aerobic spore forming Gram-positive bacteria that can not be classified asbelonging to one of the three other genera

Chapter 1

4

within the Bacillaceae family. The Bacillusspecies are widely distributed in soil and water,and certain strains tolerate high temperaturesand extreme pH values. Most species areharmless to humans and animals and have beenused in several traditional food fermentations,including the production in Japan of natto fromsoybeans. Only a few pathogens are known,including B. anthracis, the causative agent ofanthrax (5), B. cereus, which causes foodpoisoning and several insect pathogens of whichB. thuringiensis is the most well known (6). Thegenus Bacillus is an important source ofcommercial enzymes, such as cellulases,lipases, starch degrading enzymes and proteases(7). The low level of reported incidence of

pathogenicity of B. subtilis and the widespreaduse of its products and those of its closerelatives in the food, beverage, and detergentindustries have resulted in the granting ofGRAS (generally regarded as safe) status to B.subtilis by the U.S. Food and DrugAdministration.

The genetics and physiology of B.subtilis (8), considered to be the modelorganism for bacilli in general, are welldeveloped. Natural competence is one of severalpost-exponential phase phenomena that are acharacteristic of this bacterium (9).Transformation of competent B. subtilis wasfirst described in 1958 by Spizizen (10), andseveral reviews dealing with genetictransformation exist (11-14). Maximalcompetence develops shortly after the transitionfrom exponential to stationary growth phase,and high cell densities promote the initiation ofcompetence development via a quorum-sensingmechanism in which secreted oligopeptides areinvolved (15). In the presence of polyethylene

glycol, protoplasts of bacilli can be stabilizedand incorporate DNA from the medium. Aftersubsequent cell wall regeneration, transformedcells can be selected (16). Plasmidtransformation systems for Bacillus wereoriginally developed from plasmids of otherGram-positive bacteria, such as Staphylococcusaureus and later Lactococcus lactis (14). More

Figure 1.1. Contribution of various types of enzymes to the totalworldwide enzyme sales. 1% corresponds to approximately $ 10 million. Theproteases (shaded sections) contribute approximately 59% of total enzymesales of $1 billion.

Introduction

5

recently, plasmids based on cryptic Bacillusplasmids have been developed as well (17).

The availability of a wide variety ofgenetic tools and the recent sequencing of thecomplete genome (18) makes B. subtilis amongthe best-understood microorganisms (19). Inaddition, the protein secretion pathways of thisorganism have been elucidated in considerabledetail; for reviews see (20-22). The ability ofmany Bacillus species to secrete high levels ofproteins, both of homologous and heterologousnature, has made these bacteria of considerableimportance for biotechnological applications(7).

The current estimated value of theworldwide sales of industrial enzymes amountsto $ 1 billion, 59% of which is accounted for byproteases (23)(Fig. 1.1). The two main types ofproteases secreted by Bacillus are alkalineproteases (subtilisin, EC 3.4.21.62) and neutralproteases (thermolysin, EC 3.4.24.27).Subtilisins account for approximately 30% oftotal enzyme sales (7). Amylases, mainly fromBacillus amyloliquefaciens, account for another18% (24). Thus enzymes from Bacillus speciesgenerate more than 48% of total enzyme sales.

Thermolysin is used in diverseapplications in the leather industry, in bakingand in breweries (24, 25). Although proteasesare hydrolytic enzymes, they also catalyze thereverse reaction resulting in peptide synthesis.This applies in particular to the synthesis of theartificial sweetener aspartame, a dipeptidecomposed of L-aspartic acid and the methylester of L-phenylalanine, by thermolysinvariants (26, 27). DSM is one of the majorindustrial producers of aspartame.

The subtilisins and thermolysin-likeproteases (TLPs) together constitute more than95% of the extracellular protease content andare called major proteases (28). Subtilisinconstitutes approximately 15%, and the TLPsapproximately 80% of all proteolytic activityexpressed by Bacillus (29). These two proteases

are produced mainly at the end of theexponential growth phase and duringsporulation (4, 28, 29). Mutant Bacilli, failing toexpress these proteases, show normal growthand sporulation in artificial growth media (30).In nature, these proteases are thought to liberateamino acids and small peptides from externalsources (29).

1.3 Enzyme classification and thethermolysin-like proteases.

By the late 1950's the number of knownenzymes had increased rapidly; however therewas no guiding authority coordinating thenomenclature. This situation resulted in achaotic and unintelligible enzyme nomenclature.On initiative of the International Union ofBiochemistry, in cooperation with theInternational Union of Pure and AppliedChemistry (IUPAC), a committee was formedthat created the guidelines for the currentenzyme nomenclature. Today the nomenclatureis maintained by the International Union ofBiochemistry and Molecular Biology (IUBMB)(31).

Enzymes are classified according to thetype of reaction they catalyze e.g.oxidoreductases, transferases, hydrolases,lyases, isomerases and ligases. Theclassification of peptidases, which hydrolysepeptide bonds, according to their mode of actionon substrates (Fig. 1.2) has proved moreinformative for the exopeptidases than for theendopeptidases, as may be judged from the factthat subclasses have been further subdividedonly with respect to exopeptidases (31). Sinceendopeptidases all cut within a peptidesubstrate, a further subdivision based on theirmode of action on substrates can not be made.However, Hartley (32) pointed out that fourdistinct types of catalytic mechanisms are beingused by peptidases, namely serine-, cystein-,aspartic- and metallotype mechanisms. Thecarboxypeptidases and the endopeptidases have

Chapter 1

6

been subdivided on the basis of their catalyticmechanism (Fig. 1.3).

Each enzyme has a code numberconsisting of 4 numbers, e.g. thermolysin EC3.4.24.27. The first figure is the class number.All enzymes are divided in 6 classes, class 3being the hydrolases which catalyze thehydrolytic cleavage of C-O, C-N, C-C and someother bonds, including phosphoric anhydridebonds. The second figure is the subclass.Subclass 3.4, one of the 12 current subclasses ofclass 3, consists of those hydrolases that act onpeptide bonds. The third figure usually specifiesthe nature of the substrate on which the enzymeacts. However, in the case of the peptidyl-peptide hydrolases the third figure is based onthe nature of the catalytic mechanism. Sub-subclass 3.4.24 consists of those endopeptidasesthat have water as the nucleophile and have thewater molecule coordinated by a metal ion

(metallo-endopeptidases). The fourth figure isthe serial number of an enzyme in its sub-subclass. Thus, thermolysin EC 3.4.24.27 is ahydrolase, acting on a peptide bond, belongs tothe sub-subgroup of metallo-endopeptidases,and is the 27th member of this sub-subclass (Fig.1.3).

Ideally the systematic name of apeptidase should be derived from the reactioncatalyzed. However for the majority of theendopeptidases the specificity is too complex toprovide the basis for a name. In such cases,trivial names such as trypsin and thermolysinmay serve quite well. Rawlings and Barrett (33),used the term "family" to describe a group ofpeptidases that on the basis of their primarystructure are believed to be evolutionary relatedin the sense that they have arisen by divergentevolution from a single ancestral protein. Inpractice, members of families are recognized bythe fact that each shows a statisticallysignificant relatedness in amino acid sequenceto at least one other member, either throughoutthe whole sequence or at least in the domainresponsible for catalytic activity. The system offamilies that is arrived at on the basis of primarystructures almost certainly contains several setsof families that have a common evolutionaryorigin. Proteins in these sets of families havediverged from a single ancestral protein, buthave diverged to such an extent that theirprimary structure is no longer indicative of theirrelatedness. Therefore, such proteins areaccommodated in separate families. However,indications of distant relationship such as thelinear order of the catalytic residues and thetertiary structure of proteins suggest a commonancestor. The term "clan" was introduced (33) todescribe such a group of families. As yet,however, there are no generally accepted,objective methods by which to decide whetherthese similarities truly reflect divergentrelationships as opposed to convergence ofstructures under evolutionary pressure.

Figure 1.2. Classification of peptidases by the type ofreaction catalyzed. Indicated are the cleavage sites onthe various substrates. Filled circles are the residuescomprising blocks of one, two, or three terminal aminoacids that are cleaved off by these enzymes. The trianglesindicate the N- and C- terminal modification of peptidesthat provide substrates for some of the omega peptidases.Further subdivisions of the carboxypeptidases andendopeptidases have been made on the basis of catalytictype, as shown in Fig. 3.

Introduction

7

The M4 family (34) is represented bythermolysin (EC 3.4.24.27) as its prototype andconsists of secreted eubacterial endopeptidasesfrom both Gram-positive and Gram-negative

sources. Thermolysin is produced by thethermophilic Bacillus thermoproteolyticus (35).The neutral proteases, or thermolysin-likeproteases (TLPs) are inhibited by specific zinc

Figure 1.3. IUPAC enzyme classification. The peptidases, a subclass of hydrolases, are subdivided into exo- andendopeptidases, based on their mode of action on substrates. The carboxy- and exopeptidases have been furthersubdivided according to their catalytic type. Due to the establishment of the primary structure of a large number ofpeptidases it has become possible to further subdivide the peptidases based on evolutionary relationship. Themetalloendopeptidases are divided in 13 clans. Clan MA consists of 20 families, one of which is the M4 orthermolysin family. Only 6 peptidases of this family have been provided with their own EC number (see text fordetails).

Chapter 1

8

chelators, such as 1.10-phenantroline and EDTAand have their pH optimum mainly at neutralpH (36). The M4 family currently consists ofapproximately 38 members, six of which havetheir own EC number. The M4 family in turn isone of many families that form the clan MA.The MA clan is characterized by a waternucleophile in the form of water bound by asingle zinc ion ligated to two histidine residues,within the motif HExxH, and Glu, His or Asp asthe catalytic unit. Other families in the MA claninclude M34, with anthrax lethal factor as itsprototype and M13, with neprilysin as itsprototype. The MA clan together with otherclans that can be distinguished on the basis oftheir catalytic residues and the metal ionsinvolved in catalysis, form the sub-subclass ofmetallo endopeptidases EC 3.4.24. (Fig. 1.3).The primary sequence (37) and structure ofthermolysin (38) were published as early as1972. All TLPs bind two calcium ions in adouble calcium binding site, whereas the morestable TLPs bind two additional calcium ions intwo separate single calcium binding sites (39).

Subtilisin (EC 3.4.21.62), member of thesub-subgroup of serine endopeptidases, is theprototype of family S8A (40). This family inturn is part of clan SB. The SB clan ischaracterized by a serine nucleophile and has itscatalytic residues in the order Asp, His, Ser inthe primary amino acid sequence. In contrast tothe TLPs of family M4, subtilisins are inhibitedby phenylmethylsulfonylfluoride (PMSF) anddi-isopropylfluorophosphate (DIPF) and havetheir pH optimum mainly at alkaline pH (4, 41-43).

1.4 Enzyme catalysis.During the last few decades many key

questions in the life sciences have been solved.However, the origin of the catalytic power ofenzymes is still controversial (44). Although asearly as 1946, Pauling already postulated (45)that an enzyme might be complementary to the

transition state of a reaction and accelerate thereaction by binding the transition state andlowering the energy of activation, thequantitation of the physico chemical propertiesinvolved is still problematic. The catalyticpower of enzymes, that is the acceleration ofchemical reactions, should be explained by thecontribution that various enzyme-substrateinteractions have to lowering the energy of thetransition state without the need to invokespecial enzymatic secrets i.e. a special force.

The catalytic power of an enzyme issimple in its definition (44), namely the rate ofthe catalyzed reaction divided by the rate of theuncatalyzed reaction. The simplicity of thisdefinition is in contrast with the problems ofmeasuring the rate enhancement. A few of theproblems include (a) the frequent lack of ameasurable uncatalyzed reaction, (b)comparison of a pseudo-first order enzymaticreaction to a second order simple catalyst (suchas OH-), and (c) the automatic exclusion ofproximity effects if an intramolecular catalyst isused for comparison. Nevertheless, the rateenhancement of chymotrypsin and triose-phosphate isomerase are estimated at 108 to1012-fold (44).

Most of the current theories includePaulings suggestion that an enzyme might becomplementary to the transition state of areaction, enhancing the reaction by binding thetransition state and lowering the energy ofactivation. Some aspect of entropy is usuallyincorporated, although it can take many forms.Ideas of orbital steering (46, 47), solvation (48),low barrier hydrogen bonds (49), electrostatics,and pre-organized active sites all incorporateentropic factors. The second problem inexplaining the catalytic power of enzymes is thedetermination of the magnitude of thecontributions to the rate enhancement that thesedifferent effects have.

One of the most popular models toexplain the catalytic power of enzymes is the

Introduction

9

model of the pre organized active sites (50). Inpolar solvents half of the energy gained fromcharge-dipole interactions between a substrateand the solvent is spent on changing the dipole-dipole interaction needed to stabilize thetransition state. In proteins however, internalwater molecules and ionized residues arealready partially oriented toward the transitionstate charge center. Thus, the reorganization

energy is lower and the enzyme is a moreefficient catalyst than the solvent. One of theimplications of this model is that electrostaticinteractions within the active site areresponsible for the majority of the stabilizationof the transition state due to the pre-organizedpolar environment.

Figure 1.4. Abbreviated mechanism of thermolysin catalysis, as advanced byMatthews and co-workers (61, 62). The Zn2+-bound water molecule is displacedtowards Glu143 upon substrate binding. The tetrahedral intermediate, formed after anucleophilic attack of the displaced water, coordinates to the Zn2+, forming a tetrahedraltransition state. His231 acts as proton donor in the subsequent cleavage step. See text forfurther details. The figure was reproduced from (63).

Figure 1.5. Summary of thermolysin catalysis, as advanced by Mock and co-workers(63, 64). The Zn2+-bound water molecule is displaced upon substrate binding, and isactivated by His231 to perform a nucleophilic attack. His231 performs a crucial protondonation in the subsequent cleavage step. This figure was reproduced from (63).

Chapter 1

10

1.5 Catalysis in thermolysin-like proteases.The amino acid sequences of several

TLPs have been determined [see (51), or theMerops data base at http://www.merops.co.uk/merops/famcards/m4.htm], and the three-dimensional structure of TLPs isolated fromseveral bacteria have been solved i.e. Bacillusthermoproteolyticus (52), Bacillus cereus (53,54), Pseudomonas aeruginosa (55) andStaphylococcus aureus (56). TLPs consist of anα-helical C-terminal domain and an N-terminaldomain mainly consisting of β-strands. Thedomains are connected by a central α-helix. Theactive site is located in the cleft between thesetwo domains, and the catalytically essential Zn2+

ion is located at the bottom of this cleft. TheZn2+ ion is co-ordinated by His142, His146 andGlu166.

In a significant number of publishedstructures in which TLN was co-crystallisedwith inhibitors (52, 57-61), the residuesinvolved in catalysis could be identified.Matthews and co-workers proposed amechanism, presented in Fig. 1.4, in which thewater molecule bound to the Zn2+ is displaced toGlu143 upon substrate binding. The watermolecule is then activated by Glu143 andperforms a nucleophilic attack on the carbonylgroup in the scissile bond. The carboxyl groupis polarised by Tyr157, His231 and the Zn2+.The nucleophilic attack leads to a tetrahedralintermediate which co-ordinates the Zn2+ withboth oxygens. The tetrahedral intermediate isstabilised by Glu143, Tyr157 and His231. Inthis mechanism, His 231 acts as proton donor inthe subsequent cleavage step and the side chainof Asn112 and the carbonyl oxygen of residue113 stabilises the newly formed amino group(62).

This mechanism was questioned byMock and co-workers (63, 64). Theirexperiments suggested that the pH-dependenceof kcat and Km was incompatible with the role of

Glu143 and His231. Instead, Mock et al.proposed a reverse protonation mechanism inwhich the acidic limb of the pH profile isdetermined by a water molecule or hydroxideion, bound to the Zn2+ ion, whereas the basiclimb is determined by His231 (Fig. 1.5).According to Mock et al. the kcat increases withincreasing pH and has a pKa of 8.3. This wouldmean that the kcat depends on a deprotonatedHis231. Therefore, His231 can not be a protondonor but must be a proton acceptor. Mock etal. therefore proposed that TLPs follow areverse protonation mechanism.

Site-directed mutagenesis studies haveconfirmed the importance of various residues inthe active site. Beaumont et al. (65) showed thatmutating His231 in thermolysin resulted in adrastic decrease in activity and a greatly reducedpH dependence of activity in the alkaline range.Toma et al (66) confirmed the importance ofGlu143 and His231 in B. subtilis TLP (TLP-sub) by showing that mutation of these residuesresulted in a drastic reduction of activity.However, results from mutagenesis studiesseem to favour the less critical role of theHis231, as proposed by Matthews and co-workers (62, 65).

1.6 Changing the pH-activity profile ofthermolysin-like proteases.

The pH-profile of an enzyme is a ratherbroad term, which can have several meanings.In this thesis the following definitions are used:• pH-performance profile: The specific

activity measured by a given assay as afunction of pH under a given set ofconditions.

• pH-activity profile (kcat-profile or kcat/Km-profile): The kcat or kcat/Km as a function ofpH under a given set of conditions,excluding any effects from stability on theseparameters.

Introduction

11

• pH-stability profile: The stability of theenzyme as function of pH under a given setof conditions.

The pH-performance profile (which isoften called the pH-profile) of an enzyme is, infact, a combination of the pH-activity profileand the pH-stability profile under the conditionswhere the assay is performed. To be able todetermine the pH-activity profile, assayconditions have to be used that do not affect thepH-stability.

The use of Furylacryloyl (Fa) substrates,particularly Fa-Gly-Leu-Ala (FaGLA), allowthe determination of initial rates of substratedegradation. As long as the initial rate shows alinear relation between time and substrateconversion, it may be assumed that the enzymeis stable during the time of the assay and,therefore, that the pH stability remainsunchanged. The effects measured are thereforechanges in the pH-activity profile.

The dependence of the Michaelis-Menten parameters on the pKa values of theactive site residues in either the substrate-boundor substrate-free form can be derived usingkinetic equations (67). The kcat depends only onthe pKa values of the active site groups whenthe substrate is bound. The Km depends on thepKa values of both the substrate-bound andsubstrate-free form of the enzyme. Informationon the pKa values in the substrate-free form ofthe enzyme is obtained by measuring kcat/Km.

To change the pH-activity profile, thepKa values of the active site residues have to bechanged. The pKa values of titratable groups inproteins can be changed by altering theirenvironment. Three possible changes in theenvironment of a titratable group and theireffects on a positively and negatively chargedgroup are shown in Fig. 1.6. The change in pKaupon placing an acid in a negative, positive orhydrophobic environment can be summarized inthe following way;

Placing a negative charge close to theacid will give an unfavourable interaction withthe negative charge of the charged form of theacid. Compared to the situation in water thismeans that the charged state of the acidbecomes less favourable than the neutral state.The pKa value of a titratable group is the pHvalue where the group is half-protonated.Placing a negative charge close to an acid willmake an acid keep its proton longer uponraising the pH, compared to the situation inwater, and the pKa of the acid will, therefore, beelevated. Placing a positive charge next to theacid will give a favourable interaction betweenthe negative charge of the deprotonated acid.Thus the acid will loose its proton sooner withincreasing pH than it would have done so in

Figure 1.6. Environmental effects on the pKa. Theeffect on the pKa value of an acid (exemplified by aglutamic acid in panel A) and on a base (exemplified by alysine in panel B). The effect on the pKa values ofinserting a charged residue nearby is the same for the acidand the base, namely an upward shift in the pKa upon theinsertion of a negative charge, and a downward shift inthe pKa upon insertion of a positive charge. Transfer ofan acid to a hydrophobic environment gives an upwardshift in the pKa value, whereas a hydrophobicenvironment causes a base to shift its pKa valuedownwards (67).

Chapter 1

12

water. Consequently, the pKa value of an acidwould be lowered when a positive charge isplaced in its close proximity.

Generally, it is unfavourable for acharged residue to reside in a hydrophobicenvironment as compared to residing in water.Placing an acid in a hydrophobic environmentwill therefore have the same effect as placing itin a negative environment. The pKa value of theacid will therefore be elevated in a hydrophobicenvironment. The effect of the environment onthe pKa of a base can be deduced in the sameway as for an acid. For a base, however, itshould be noted that the charged form ispredominant at low pH values, whereas for theacid the charged form is dominant at high pHvalues.

Although it is well established that pKavalues can be modified by adjacent residues(68), rational modification of the pH-performance profile, or pH-optimum, hasproven to be difficult. In the most successfulstudies, natural variants with a different pH-optimum have been used to design mutationsclose to the active site (69, 70). This approach islimited to those cases where well characterised,highly homologous proteins with a different pHoptimum are available. Even then success is notguaranteed, as mutations in and close to theactive site often produce strong negative effectson the activity (71-73). An alternative approachto mutations close to the active site is themodification of the surface charge of theenzyme (72-74). Since the electrostaticinteraction between a charged group and atitratable group is a long-range interaction,mutations outside the active site should be ableto influence the pKa values of the active siteresidues. In this respect, Russell et.al. (74) haveshown that mutating residues as far as 15Å fromthe active site of subtilisin caused a shift in thepKa values of the active site residues ofapproximately 0.4 pH units. However, even the

complete reversal of the surface charge does notnecessarily have an effect on catalysis (75).

1.7 Engineering thermal stability ofthermolysin-like proteases.

For an extensive review on theengineering of the thermal stability determinantsof TLPs see (76). Bacillus strains display largedifferences in optimum growth temperature andthe stabilities of their TLPs vary accordingly(77). The thermal stability of the thermolysin-like protease from B. stearothermophilus (TLP-ste) is considerably lower than that ofthermolysin, which is the most stable TLP.Initial protein engineering studies of TLP-stewere aimed at stabilising the enzyme bymutations designed on the basis of generalprinciples of protein stability (78). On the basisof these results, it was suggested that thermalinactivation of TLP-ste is governed by localunfolding processes that involve only parts ofthe molecule. Mutations with large effects onstability were located in or near a surface-loopin the N-terminal domain, spanning residues 56through 69. Combining 5 stabilising TLP-ste�thermolysin mutations (A4T, T56A, G58A,T63F, A69A), four of which are located in thestability determining surface loop, resulted in anenzyme that was considerably more stable thanthermolysin itself (79).

An important consequence of the factthat stability-determining unfolding processes inTLP-ste have a local character is that mutationaleffects may display extreme non-additivity (80).To illustrate this, Vriend et al. (81) created fourpseudo wild-types of TLP-ste in which a secondunfolding region had been created by mutationsin the C-terminal domain of the enzyme. Aftermaking these pseudo wild-types, the effects ofstabilising the two regions individually orsimultaneously were studied. The resultsobtained with these mutants show the ”enoughis enough” effect (80). This effect means that itdoes not help to stabilise a region of the protein

Introduction

13

once this region has become so stable that itsunfolding no longer contributes to the overallthermal inactivation process. It is interesting tonote that mutations in the hydrophobic core ofthe C-terminal domain that had surprisinglymarginal effects on the stability of wild-typeTLP-ste, became important once the N-terminalunfolding region had been considerablystabilised. In other words, the contribution of asecond unfolding process (involving thehydrophobic core of the C-terminal domain) tothe overall rate of thermal inactivation becomesnoticeable after the contribution of the othermajor unfolding process (involving the 56-69region) has been reduced by stabilisingmutations.

The most spectacular example of adesigned stabilising mutation in the 56-69region in TLP-ste was an engineered disulphidebridge between residues 8 and 60 whichincreased the T50 by as much as 16.7 degrees(82). This result contrasts with the rathermarginal stability effects that were obtainedupon introducing a variety of designeddisulphide bridges in the broad-specificityprotease subtilisin (83). Eijsink et al. (76)propose that the lack of success of theengineered disulphide bridges in subtilisin is atleast partly due to the fact that these bridgeswere introduced in to regions of the proteasethat do not play a dominant role in stability-determining local unfolding processes.

Combination of the five stabilising TLP-ste�thermolysin mutations (A4T, T56A, G58A,T63F, A69P) with the designed mutations S65P,G8C, and N60C yielded one of the most stableproteins ever obtained by protein engineering(84, 85). This 8-fold variant had a half-life of170 minutes at 100 oC and was namedboilysin™. Boilysin was stable for at least 24hours at 90 degrees, a temperature at which thehalf-life of the original enzyme (wild-type TLP-ste) was approximately one minute. It wasshown that, in contrast to wild-type TLP-ste,

boilysin tolerates considerable amounts of urea,guanidinium-HCl and SDS. For example, theenzyme retained approximately 60 percent of itsactivity in the presence of 5 M urea and 40 % ofits activity in the presence of 1 % (wt/vol) SDS(84).

1.8 Engineering hinge bending inthermolysin-like proteases.

Many proteins undergo hinge-bendingmotions during catalysis (86-90). Hinge-bending is the motion of two domains of anenzyme around a hinge point or hinge axis.Through this hinge movement one domain of anenzyme can close onto another to isolate asubstrate from the environment. In several casesglycine residues have been shown crucial forproviding a protein with the necessary (hinge-bending) flexibility (54, 89, 90). Van Aalten etal. (91) showed that mutation of a glycineresidue in the proposed hinge region in retinolbinding protein indeed dramatically reducedretinol binding.

Originally, the crystal structure ofthermolysin was supposed to be that of the pureenzyme (92, 93). When the crystal structure ofB. cereus TLP (TLP-cer) became available, itwas noticed that the active site cleft of thisenzyme was more open than that in thermolysin,resulting from a hinge-bending between the N-and C-terminal domains (54, 86). Puzzled bythis observation, Holland et al. (86) re-examined the original crystallographic data forthermolysin and TLP-cer. They concluded thatthe structure of thermolysin was not that of thefree enzyme, but of the enzyme containing adipeptide in the active site. This peptide was notpresent in the TLP-cer structure. Theseobservations suggested that a hinge-bendingmotion is part of the catalytic mechanism ofTLPs, and that substrate binding yields a closureof the active site (86). Interestingly, a hinge-bending motion quite similar to the oneproposed by Holland et al. was observed when

Chapter 1

14

concerted motions in thermolysin were studiedusing so-called 'essential dynamics' analysis ofmolecular dynamics simulations of thermolysin(89). Originally, Stark et al. (54) suggested thatthe hinge-bending region in TLP-cer andthermolysin is located around glycines 135 and136. This is an attractive proposal, since itplaces the hinge at the beginning of the α-helixconnecting the two domains between whichhinge-bending occurs. In later studies, Hollandet al. proposed a more complex scheme, inwhich bending of the N-terminal α-helix,around Gly78, plays a prominent role. On thebasis of their molecular dynamics simulations,Van Aalten et al. suggested the hinge region inthermolysin to be at both ends of the α-helixconnecting the N- and C-terminal domain(residues 135 and 136 are on the N-terminal endof the connecting α-helix).

The alignment of several TLPs [see (54)or http://www.merops.co.uk/merops/famcards/m4.htm] shows that Gly78 and several glycinesin the α-helix connecting the two domains areconserved in certain groups of TLPs. Residue135 is the most conserved glycine; otherglycines are less well conserved, but acorrelation exists between the disappearance of

certain glycines and the appearance of others.For example, a glycine at 147 is conserved inthose TLPs that have no glycine at position 136.All these glycine residues (78, 135, 136, 141,147, 154) have dihedral angles that arecompatible with some non-glycine amino acidresidues and therefore, are unlikely to representresidues exclusively required for the 3Dstructure of the enzyme. Rather they may berequired for hinge-bending.

1.9 Engineering substrate specificity andactivity.

Rational design of substrate specificity isone of the main goals of protein engineering.Exploitation of enzymes in industry would befacilitated by the ability to rationally modify thesubstrate specificity of an enzyme.Consequently, an extensive body of literatureexists on engineering the substrate specificity ofproteases. The subsite and substratenomenclature regarding specificity issummarized in Fig. 1.7 (94). Three examples ofspecificity determinants are discussed below.

The first and most common example isthe engineering of the substrate binding pocketsto change the substrate specificity. Mei et al.

Figure 1.7. Subsite and substrate nomenclature of proteases. The amino acids of the substrate are countedfrom the cleavage site toward the N-terminus as P1, P2 etc, and towards the C-terminus as P1', P2' etc. Thecorresponding binding pockets on the enzyme are called S1, S2 and S1' and S2'.

Introduction

15

(95) replaced glycines in the S1 subsite ofsubtilisin YaB by larger residues such as alanineand valine. This resulted in an increase inactivity towards substrates with a P1 Ala and asharp decrease in activity towards substrateswith a P1 Phe or Leu. Many other examplesexist in which the preference for largehydrophobic substrates was diminished byreducing the substrate binding pocket sizethrough the replacement of small binding pocketresidues by larger residues (95-97) or byblocking the entrance of the binding pocket(98).

The second example concerns theconversion of trypsin to chymotrypsin. One ofthe most thoroughly studied and now bestunderstood systems is the conversion of trypsinto chymotrypsin and the structural basis ofsubstrate specificity in the serine proteases (99-101). Trypsin (EC 3.4.21.4) and chymotrypsin(EC 3.4.21.1) both belong to the S1 peptidasefamily and catalyze peptide bond cleavage byidentical mechanisms. A serine residue acts as anucleophile and the catalytic residues are in theorder His, Asp, Ser in the primary sequence.Both enzymes are endopeptidases and possessvery similar tertiary structures consisting of twojuxtaposed six stranded β-barrel domains (102,103). The substrate specificity of trypsin,expressed in relative kcat/Km values, is nearly106-fold higher for P1 Arg or Lys containingsubstrates compared to the activity towardsanalogous P1 Phe containing substrates.Conversely, chymotrypsin favours peptidesubstrates possessing Trp, Tyr and Phe at the P1

position, with an overall specificity relative toP1 Lys substrates of up to 104-fold.

Since the structures of the S1 subsites ofthe two enzymes are very similar, the differencein substrate specificity was thought to be asimple property of the local electrostaticenvironment. However, replacement of theprimary binding determinant Asp189 of trypsinwith the analogous Ser189 of chymotrypsin

failed to convert the specificity but, instead,resulted in a poorly performing nonspecificprotease (104). Conversion of trypsin to achymotrypsin-like protease required thesubstitution of four residues in the S1 subsitetogether with the exchange of two adjacentsurface loops, which do not directly contact thesubstrate (105). Inspection of the crystalstructures of the wild-type trypsin andchymotrypsin and those of several mutants,revealed the specificity determinants involved(99). The conserved Gly216, which contacts theP3 residue in both trypsin and chymotrypsin,turned out to be crucial for correct positioningof the substrate in the active site. The differentstructures of the surface loops in trypsin andchymotrypsin maintain Gly216 in distinctconformations, enabling this residue to functionas a specificity determinant despite beingconserved in both proteases.

The study of the trypsin-chymotrypsinsystem has led to a definition of two types ofspecificity determinants (99); primaryspecificity determinants encompassing aminoacids that directly contact the substrate, andsecondary specificity determinants which aremore distantly located elements in the protein.The secondary determinants can act throughvarious mechanisms such as influencing theconformation of primary determinants, as in thecase of Gly216 in trypsin and chymotrypsin, orby modulating the degree of flexibility in thesubstrate binding site. Examples of the latter canbe found in elastase (106, 107) and coenzyme Atransferase (108). The existence of secondaryspecificity determinants imply that substratespecificity is not necessarily determined by alimited set of amino acids in the substratebinding pockets. Instead, substrate specificitycan be a globally distributed propertydetermined by a large part of the protein fold.

The third example is another example ofa specificity determinant which is not located ina subsite. This example relates to the S10 family

Introduction

3

Introduction

1. Introduction.1.1 General.

Enzymes offer many advantages overtraditional chemical catalysts. They are clean,often cheap, and renewable. However, fullexploitation of enzymes in industry requires themodification of enzymes in such a way that theyperform a desired reaction under a set ofspecific conditions. The problems that have tobe solved to be able to rationally modifyenzymes are diverse and relate to questions suchas: why are proteins folded the way they are,which structural features determine proteinstability, how does enzymatic catalysis work,and which structural elements control thespecificity of an enzyme? Protein engineering isthe multi-disciplinary approach to answer thesequestions by studying the structure-functionrelationships of proteins. Once these structure-function relationships are known, the obtainedknowledge can be exploited to endow proteinswith changed properties. Since the breakthroughof recombinant DNA technology tremendousprogress has been made in solving structure-function relationships. For example, theunderstanding of thermostability has led toalgorithms that can predict which changes in theprimary structure are likely to improve thethermostability of a protein (1). Proteinengineering could eventually enable the de novodesign of virtually any enzymatic property.

However, fully de novo design of anenzyme is still far away, and many questions onthe structure-function relationships of enzymesremain unanswered. One of the most importantsteps of the last decade in exploiting enzymes isundoubtedly the development of molecular

breeding strategies (2), in which gene splicingand random mutagenesis are combined to yieldenzymes with novel or strongly improvedproperties. Molecular breeding and otherrandom mutagenesis methods (3) are greatlyfacilitated by the advent of robotics, enablingthe handling and screening of tens of thousandsof mutants.

However impressive the results withrandom mutagenesis methods are in yieldingnovel or improved biocatalysts, they do notdirectly contribute to the knowledge of howproteins work. Reverse engineering, that isobtaining an enzyme with a novel functionalitythrough random mutagenesis and reverting itstep by step to the wild-type lacking thisfunctionality, would help in revealingcomplicated structure function-relationshipswhile at the same time yielding many newbiocatalysts.

This thesis describes the developmentand use of protein engineering technologies tochange the properties of thermolysin-likeproteases produced by various Bacillus species.

1.2 The exploitation of the genus Bacilluswith respect to industrially importantenzymes.

The Gram-positive family ofBacillaceae contains four aerobic, endospore-forming genera i.e. Thermoactinomyces,Sporosarcina, Sporolactobacillus and Bacillus(4). Compared to the three other genera, thegenus Bacillus is quite heterogeneous: itconsists of all the aerobic spore forming Gram-positive bacteria that can not be classified asbelonging to one of the three other genera

Chapter 1

4

within the Bacillaceae family. The Bacillusspecies are widely distributed in soil and water,and certain strains tolerate high temperaturesand extreme pH values. Most species areharmless to humans and animals and have beenused in several traditional food fermentations,including the production in Japan of natto fromsoybeans. Only a few pathogens are known,including B. anthracis, the causative agent ofanthrax (5), B. cereus, which causes foodpoisoning and several insect pathogens of whichB. thuringiensis is the most well known (6). Thegenus Bacillus is an important source ofcommercial enzymes, such as cellulases,lipases, starch degrading enzymes and proteases(7). The low level of reported incidence of

pathogenicity of B. subtilis and the widespreaduse of its products and those of its closerelatives in the food, beverage, and detergentindustries have resulted in the granting ofGRAS (generally regarded as safe) status to B.subtilis by the U.S. Food and DrugAdministration.

The genetics and physiology of B.subtilis (8), considered to be the modelorganism for bacilli in general, are welldeveloped. Natural competence is one of severalpost-exponential phase phenomena that are acharacteristic of this bacterium (9).Transformation of competent B. subtilis wasfirst described in 1958 by Spizizen (10), andseveral reviews dealing with genetictransformation exist (11-14). Maximalcompetence develops shortly after the transitionfrom exponential to stationary growth phase,and high cell densities promote the initiation ofcompetence development via a quorum-sensingmechanism in which secreted oligopeptides areinvolved (15). In the presence of polyethylene

glycol, protoplasts of bacilli can be stabilizedand incorporate DNA from the medium. Aftersubsequent cell wall regeneration, transformedcells can be selected (16). Plasmidtransformation systems for Bacillus wereoriginally developed from plasmids of otherGram-positive bacteria, such as Staphylococcusaureus and later Lactococcus lactis (14). More

Figure 1.1. Contribution of various types of enzymes to the totalworldwide enzyme sales. 1% corresponds to approximately $ 10 million. Theproteases (shaded sections) contribute approximately 59% of total enzymesales of $1 billion.

Introduction

5

recently, plasmids based on cryptic Bacillusplasmids have been developed as well (17).

The availability of a wide variety ofgenetic tools and the recent sequencing of thecomplete genome (18) makes B. subtilis amongthe best-understood microorganisms (19). Inaddition, the protein secretion pathways of thisorganism have been elucidated in considerabledetail; for reviews see (20-22). The ability ofmany Bacillus species to secrete high levels ofproteins, both of homologous and heterologousnature, has made these bacteria of considerableimportance for biotechnological applications(7).

The current estimated value of theworldwide sales of industrial enzymes amountsto $ 1 billion, 59% of which is accounted for byproteases (23)(Fig. 1.1). The two main types ofproteases secreted by Bacillus are alkalineproteases (subtilisin, EC 3.4.21.62) and neutralproteases (thermolysin, EC 3.4.24.27).Subtilisins account for approximately 30% oftotal enzyme sales (7). Amylases, mainly fromBacillus amyloliquefaciens, account for another18% (24). Thus enzymes from Bacillus speciesgenerate more than 48% of total enzyme sales.

Thermolysin is used in diverseapplications in the leather industry, in bakingand in breweries (24, 25). Although proteasesare hydrolytic enzymes, they also catalyze thereverse reaction resulting in peptide synthesis.This applies in particular to the synthesis of theartificial sweetener aspartame, a dipeptidecomposed of L-aspartic acid and the methylester of L-phenylalanine, by thermolysinvariants (26, 27). DSM is one of the majorindustrial producers of aspartame.

The subtilisins and thermolysin-likeproteases (TLPs) together constitute more than95% of the extracellular protease content andare called major proteases (28). Subtilisinconstitutes approximately 15%, and the TLPsapproximately 80% of all proteolytic activityexpressed by Bacillus (29). These two proteases

are produced mainly at the end of theexponential growth phase and duringsporulation (4, 28, 29). Mutant Bacilli, failing toexpress these proteases, show normal growthand sporulation in artificial growth media (30).In nature, these proteases are thought to liberateamino acids and small peptides from externalsources (29).

1.3 Enzyme classification and thethermolysin-like proteases.

By the late 1950's the number of knownenzymes had increased rapidly; however therewas no guiding authority coordinating thenomenclature. This situation resulted in achaotic and unintelligible enzyme nomenclature.On initiative of the International Union ofBiochemistry, in cooperation with theInternational Union of Pure and AppliedChemistry (IUPAC), a committee was formedthat created the guidelines for the currentenzyme nomenclature. Today the nomenclatureis maintained by the International Union ofBiochemistry and Molecular Biology (IUBMB)(31).

Enzymes are classified according to thetype of reaction they catalyze e.g.oxidoreductases, transferases, hydrolases,lyases, isomerases and ligases. Theclassification of peptidases, which hydrolysepeptide bonds, according to their mode of actionon substrates (Fig. 1.2) has proved moreinformative for the exopeptidases than for theendopeptidases, as may be judged from the factthat subclasses have been further subdividedonly with respect to exopeptidases (31). Sinceendopeptidases all cut within a peptidesubstrate, a further subdivision based on theirmode of action on substrates can not be made.However, Hartley (32) pointed out that fourdistinct types of catalytic mechanisms are beingused by peptidases, namely serine-, cystein-,aspartic- and metallotype mechanisms. Thecarboxypeptidases and the endopeptidases have

Chapter 1

6

been subdivided on the basis of their catalyticmechanism (Fig. 1.3).

Each enzyme has a code numberconsisting of 4 numbers, e.g. thermolysin EC3.4.24.27. The first figure is the class number.All enzymes are divided in 6 classes, class 3being the hydrolases which catalyze thehydrolytic cleavage of C-O, C-N, C-C and someother bonds, including phosphoric anhydridebonds. The second figure is the subclass.Subclass 3.4, one of the 12 current subclasses ofclass 3, consists of those hydrolases that act onpeptide bonds. The third figure usually specifiesthe nature of the substrate on which the enzymeacts. However, in the case of the peptidyl-peptide hydrolases the third figure is based onthe nature of the catalytic mechanism. Sub-subclass 3.4.24 consists of those endopeptidasesthat have water as the nucleophile and have thewater molecule coordinated by a metal ion

(metallo-endopeptidases). The fourth figure isthe serial number of an enzyme in its sub-subclass. Thus, thermolysin EC 3.4.24.27 is ahydrolase, acting on a peptide bond, belongs tothe sub-subgroup of metallo-endopeptidases,and is the 27th member of this sub-subclass (Fig.1.3).

Ideally the systematic name of apeptidase should be derived from the reactioncatalyzed. However for the majority of theendopeptidases the specificity is too complex toprovide the basis for a name. In such cases,trivial names such as trypsin and thermolysinmay serve quite well. Rawlings and Barrett (33),used the term "family" to describe a group ofpeptidases that on the basis of their primarystructure are believed to be evolutionary relatedin the sense that they have arisen by divergentevolution from a single ancestral protein. Inpractice, members of families are recognized bythe fact that each shows a statisticallysignificant relatedness in amino acid sequenceto at least one other member, either throughoutthe whole sequence or at least in the domainresponsible for catalytic activity. The system offamilies that is arrived at on the basis of primarystructures almost certainly contains several setsof families that have a common evolutionaryorigin. Proteins in these sets of families havediverged from a single ancestral protein, buthave diverged to such an extent that theirprimary structure is no longer indicative of theirrelatedness. Therefore, such proteins areaccommodated in separate families. However,indications of distant relationship such as thelinear order of the catalytic residues and thetertiary structure of proteins suggest a commonancestor. The term "clan" was introduced (33) todescribe such a group of families. As yet,however, there are no generally accepted,objective methods by which to decide whetherthese similarities truly reflect divergentrelationships as opposed to convergence ofstructures under evolutionary pressure.

Figure 1.2. Classification of peptidases by the type ofreaction catalyzed. Indicated are the cleavage sites onthe various substrates. Filled circles are the residuescomprising blocks of one, two, or three terminal aminoacids that are cleaved off by these enzymes. The trianglesindicate the N- and C- terminal modification of peptidesthat provide substrates for some of the omega peptidases.Further subdivisions of the carboxypeptidases andendopeptidases have been made on the basis of catalytictype, as shown in Fig. 3.

Introduction

7

The M4 family (34) is represented bythermolysin (EC 3.4.24.27) as its prototype andconsists of secreted eubacterial endopeptidasesfrom both Gram-positive and Gram-negative

sources. Thermolysin is produced by thethermophilic Bacillus thermoproteolyticus (35).The neutral proteases, or thermolysin-likeproteases (TLPs) are inhibited by specific zinc

Figure 1.3. IUPAC enzyme classification. The peptidases, a subclass of hydrolases, are subdivided into exo- andendopeptidases, based on their mode of action on substrates. The carboxy- and exopeptidases have been furthersubdivided according to their catalytic type. Due to the establishment of the primary structure of a large number ofpeptidases it has become possible to further subdivide the peptidases based on evolutionary relationship. Themetalloendopeptidases are divided in 13 clans. Clan MA consists of 20 families, one of which is the M4 orthermolysin family. Only 6 peptidases of this family have been provided with their own EC number (see text fordetails).

Chapter 1

8

chelators, such as 1.10-phenantroline and EDTAand have their pH optimum mainly at neutralpH (36). The M4 family currently consists ofapproximately 38 members, six of which havetheir own EC number. The M4 family in turn isone of many families that form the clan MA.The MA clan is characterized by a waternucleophile in the form of water bound by asingle zinc ion ligated to two histidine residues,within the motif HExxH, and Glu, His or Asp asthe catalytic unit. Other families in the MA claninclude M34, with anthrax lethal factor as itsprototype and M13, with neprilysin as itsprototype. The MA clan together with otherclans that can be distinguished on the basis oftheir catalytic residues and the metal ionsinvolved in catalysis, form the sub-subclass ofmetallo endopeptidases EC 3.4.24. (Fig. 1.3).The primary sequence (37) and structure ofthermolysin (38) were published as early as1972. All TLPs bind two calcium ions in adouble calcium binding site, whereas the morestable TLPs bind two additional calcium ions intwo separate single calcium binding sites (39).

Subtilisin (EC 3.4.21.62), member of thesub-subgroup of serine endopeptidases, is theprototype of family S8A (40). This family inturn is part of clan SB. The SB clan ischaracterized by a serine nucleophile and has itscatalytic residues in the order Asp, His, Ser inthe primary amino acid sequence. In contrast tothe TLPs of family M4, subtilisins are inhibitedby phenylmethylsulfonylfluoride (PMSF) anddi-isopropylfluorophosphate (DIPF) and havetheir pH optimum mainly at alkaline pH (4, 41-43).

1.4 Enzyme catalysis.During the last few decades many key

questions in the life sciences have been solved.However, the origin of the catalytic power ofenzymes is still controversial (44). Although asearly as 1946, Pauling already postulated (45)that an enzyme might be complementary to the

transition state of a reaction and accelerate thereaction by binding the transition state andlowering the energy of activation, thequantitation of the physico chemical propertiesinvolved is still problematic. The catalyticpower of enzymes, that is the acceleration ofchemical reactions, should be explained by thecontribution that various enzyme-substrateinteractions have to lowering the energy of thetransition state without the need to invokespecial enzymatic secrets i.e. a special force.

The catalytic power of an enzyme issimple in its definition (44), namely the rate ofthe catalyzed reaction divided by the rate of theuncatalyzed reaction. The simplicity of thisdefinition is in contrast with the problems ofmeasuring the rate enhancement. A few of theproblems include (a) the frequent lack of ameasurable uncatalyzed reaction, (b)comparison of a pseudo-first order enzymaticreaction to a second order simple catalyst (suchas OH-), and (c) the automatic exclusion ofproximity effects if an intramolecular catalyst isused for comparison. Nevertheless, the rateenhancement of chymotrypsin and triose-phosphate isomerase are estimated at 108 to1012-fold (44).

Most of the current theories includePaulings suggestion that an enzyme might becomplementary to the transition state of areaction, enhancing the reaction by binding thetransition state and lowering the energy ofactivation. Some aspect of entropy is usuallyincorporated, although it can take many forms.Ideas of orbital steering (46, 47), solvation (48),low barrier hydrogen bonds (49), electrostatics,and pre-organized active sites all incorporateentropic factors. The second problem inexplaining the catalytic power of enzymes is thedetermination of the magnitude of thecontributions to the rate enhancement that thesedifferent effects have.

One of the most popular models toexplain the catalytic power of enzymes is the

Introduction

9

model of the pre organized active sites (50). Inpolar solvents half of the energy gained fromcharge-dipole interactions between a substrateand the solvent is spent on changing the dipole-dipole interaction needed to stabilize thetransition state. In proteins however, internalwater molecules and ionized residues arealready partially oriented toward the transitionstate charge center. Thus, the reorganization

energy is lower and the enzyme is a moreefficient catalyst than the solvent. One of theimplications of this model is that electrostaticinteractions within the active site areresponsible for the majority of the stabilizationof the transition state due to the pre-organizedpolar environment.

Figure 1.4. Abbreviated mechanism of thermolysin catalysis, as advanced byMatthews and co-workers (61, 62). The Zn2+-bound water molecule is displacedtowards Glu143 upon substrate binding. The tetrahedral intermediate, formed after anucleophilic attack of the displaced water, coordinates to the Zn2+, forming a tetrahedraltransition state. His231 acts as proton donor in the subsequent cleavage step. See text forfurther details. The figure was reproduced from (63).

Figure 1.5. Summary of thermolysin catalysis, as advanced by Mock and co-workers(63, 64). The Zn2+-bound water molecule is displaced upon substrate binding, and isactivated by His231 to perform a nucleophilic attack. His231 performs a crucial protondonation in the subsequent cleavage step. This figure was reproduced from (63).

Chapter 1

10

1.5 Catalysis in thermolysin-like proteases.The amino acid sequences of several

TLPs have been determined [see (51), or theMerops data base at http://www.merops.co.uk/merops/famcards/m4.htm], and the three-dimensional structure of TLPs isolated fromseveral bacteria have been solved i.e. Bacillusthermoproteolyticus (52), Bacillus cereus (53,54), Pseudomonas aeruginosa (55) andStaphylococcus aureus (56). TLPs consist of anα-helical C-terminal domain and an N-terminaldomain mainly consisting of β-strands. Thedomains are connected by a central α-helix. Theactive site is located in the cleft between thesetwo domains, and the catalytically essential Zn2+

ion is located at the bottom of this cleft. TheZn2+ ion is co-ordinated by His142, His146 andGlu166.

In a significant number of publishedstructures in which TLN was co-crystallisedwith inhibitors (52, 57-61), the residuesinvolved in catalysis could be identified.Matthews and co-workers proposed amechanism, presented in Fig. 1.4, in which thewater molecule bound to the Zn2+ is displaced toGlu143 upon substrate binding. The watermolecule is then activated by Glu143 andperforms a nucleophilic attack on the carbonylgroup in the scissile bond. The carboxyl groupis polarised by Tyr157, His231 and the Zn2+.The nucleophilic attack leads to a tetrahedralintermediate which co-ordinates the Zn2+ withboth oxygens. The tetrahedral intermediate isstabilised by Glu143, Tyr157 and His231. Inthis mechanism, His 231 acts as proton donor inthe subsequent cleavage step and the side chainof Asn112 and the carbonyl oxygen of residue113 stabilises the newly formed amino group(62).

This mechanism was questioned byMock and co-workers (63, 64). Theirexperiments suggested that the pH-dependenceof kcat and Km was incompatible with the role of

Glu143 and His231. Instead, Mock et al.proposed a reverse protonation mechanism inwhich the acidic limb of the pH profile isdetermined by a water molecule or hydroxideion, bound to the Zn2+ ion, whereas the basiclimb is determined by His231 (Fig. 1.5).According to Mock et al. the kcat increases withincreasing pH and has a pKa of 8.3. This wouldmean that the kcat depends on a deprotonatedHis231. Therefore, His231 can not be a protondonor but must be a proton acceptor. Mock etal. therefore proposed that TLPs follow areverse protonation mechanism.

Site-directed mutagenesis studies haveconfirmed the importance of various residues inthe active site. Beaumont et al. (65) showed thatmutating His231 in thermolysin resulted in adrastic decrease in activity and a greatly reducedpH dependence of activity in the alkaline range.Toma et al (66) confirmed the importance ofGlu143 and His231 in B. subtilis TLP (TLP-sub) by showing that mutation of these residuesresulted in a drastic reduction of activity.However, results from mutagenesis studiesseem to favour the less critical role of theHis231, as proposed by Matthews and co-workers (62, 65).

1.6 Changing the pH-activity profile ofthermolysin-like proteases.

The pH-profile of an enzyme is a ratherbroad term, which can have several meanings.In this thesis the following definitions are used:• pH-performance profile: The specific

activity measured by a given assay as afunction of pH under a given set ofconditions.

• pH-activity profile (kcat-profile or kcat/Km-profile): The kcat or kcat/Km as a function ofpH under a given set of conditions,excluding any effects from stability on theseparameters.

Introduction

11

• pH-stability profile: The stability of theenzyme as function of pH under a given setof conditions.

The pH-performance profile (which isoften called the pH-profile) of an enzyme is, infact, a combination of the pH-activity profileand the pH-stability profile under the conditionswhere the assay is performed. To be able todetermine the pH-activity profile, assayconditions have to be used that do not affect thepH-stability.

The use of Furylacryloyl (Fa) substrates,particularly Fa-Gly-Leu-Ala (FaGLA), allowthe determination of initial rates of substratedegradation. As long as the initial rate shows alinear relation between time and substrateconversion, it may be assumed that the enzymeis stable during the time of the assay and,therefore, that the pH stability remainsunchanged. The effects measured are thereforechanges in the pH-activity profile.

The dependence of the Michaelis-Menten parameters on the pKa values of theactive site residues in either the substrate-boundor substrate-free form can be derived usingkinetic equations (67). The kcat depends only onthe pKa values of the active site groups whenthe substrate is bound. The Km depends on thepKa values of both the substrate-bound andsubstrate-free form of the enzyme. Informationon the pKa values in the substrate-free form ofthe enzyme is obtained by measuring kcat/Km.

To change the pH-activity profile, thepKa values of the active site residues have to bechanged. The pKa values of titratable groups inproteins can be changed by altering theirenvironment. Three possible changes in theenvironment of a titratable group and theireffects on a positively and negatively chargedgroup are shown in Fig. 1.6. The change in pKaupon placing an acid in a negative, positive orhydrophobic environment can be summarized inthe following way;

Placing a negative charge close to theacid will give an unfavourable interaction withthe negative charge of the charged form of theacid. Compared to the situation in water thismeans that the charged state of the acidbecomes less favourable than the neutral state.The pKa value of a titratable group is the pHvalue where the group is half-protonated.Placing a negative charge close to an acid willmake an acid keep its proton longer uponraising the pH, compared to the situation inwater, and the pKa of the acid will, therefore, beelevated. Placing a positive charge next to theacid will give a favourable interaction betweenthe negative charge of the deprotonated acid.Thus the acid will loose its proton sooner withincreasing pH than it would have done so in

Figure 1.6. Environmental effects on the pKa. Theeffect on the pKa value of an acid (exemplified by aglutamic acid in panel A) and on a base (exemplified by alysine in panel B). The effect on the pKa values ofinserting a charged residue nearby is the same for the acidand the base, namely an upward shift in the pKa upon theinsertion of a negative charge, and a downward shift inthe pKa upon insertion of a positive charge. Transfer ofan acid to a hydrophobic environment gives an upwardshift in the pKa value, whereas a hydrophobicenvironment causes a base to shift its pKa valuedownwards (67).

Chapter 1

12

water. Consequently, the pKa value of an acidwould be lowered when a positive charge isplaced in its close proximity.

Generally, it is unfavourable for acharged residue to reside in a hydrophobicenvironment as compared to residing in water.Placing an acid in a hydrophobic environmentwill therefore have the same effect as placing itin a negative environment. The pKa value of theacid will therefore be elevated in a hydrophobicenvironment. The effect of the environment onthe pKa of a base can be deduced in the sameway as for an acid. For a base, however, itshould be noted that the charged form ispredominant at low pH values, whereas for theacid the charged form is dominant at high pHvalues.

Although it is well established that pKavalues can be modified by adjacent residues(68), rational modification of the pH-performance profile, or pH-optimum, hasproven to be difficult. In the most successfulstudies, natural variants with a different pH-optimum have been used to design mutationsclose to the active site (69, 70). This approach islimited to those cases where well characterised,highly homologous proteins with a different pHoptimum are available. Even then success is notguaranteed, as mutations in and close to theactive site often produce strong negative effectson the activity (71-73). An alternative approachto mutations close to the active site is themodification of the surface charge of theenzyme (72-74). Since the electrostaticinteraction between a charged group and atitratable group is a long-range interaction,mutations outside the active site should be ableto influence the pKa values of the active siteresidues. In this respect, Russell et.al. (74) haveshown that mutating residues as far as 15Å fromthe active site of subtilisin caused a shift in thepKa values of the active site residues ofapproximately 0.4 pH units. However, even the

complete reversal of the surface charge does notnecessarily have an effect on catalysis (75).

1.7 Engineering thermal stability ofthermolysin-like proteases.

For an extensive review on theengineering of the thermal stability determinantsof TLPs see (76). Bacillus strains display largedifferences in optimum growth temperature andthe stabilities of their TLPs vary accordingly(77). The thermal stability of the thermolysin-like protease from B. stearothermophilus (TLP-ste) is considerably lower than that ofthermolysin, which is the most stable TLP.Initial protein engineering studies of TLP-stewere aimed at stabilising the enzyme bymutations designed on the basis of generalprinciples of protein stability (78). On the basisof these results, it was suggested that thermalinactivation of TLP-ste is governed by localunfolding processes that involve only parts ofthe molecule. Mutations with large effects onstability were located in or near a surface-loopin the N-terminal domain, spanning residues 56through 69. Combining 5 stabilising TLP-ste�thermolysin mutations (A4T, T56A, G58A,T63F, A69A), four of which are located in thestability determining surface loop, resulted in anenzyme that was considerably more stable thanthermolysin itself (79).

An important consequence of the factthat stability-determining unfolding processes inTLP-ste have a local character is that mutationaleffects may display extreme non-additivity (80).To illustrate this, Vriend et al. (81) created fourpseudo wild-types of TLP-ste in which a secondunfolding region had been created by mutationsin the C-terminal domain of the enzyme. Aftermaking these pseudo wild-types, the effects ofstabilising the two regions individually orsimultaneously were studied. The resultsobtained with these mutants show the ”enoughis enough” effect (80). This effect means that itdoes not help to stabilise a region of the protein

Introduction

13

once this region has become so stable that itsunfolding no longer contributes to the overallthermal inactivation process. It is interesting tonote that mutations in the hydrophobic core ofthe C-terminal domain that had surprisinglymarginal effects on the stability of wild-typeTLP-ste, became important once the N-terminalunfolding region had been considerablystabilised. In other words, the contribution of asecond unfolding process (involving thehydrophobic core of the C-terminal domain) tothe overall rate of thermal inactivation becomesnoticeable after the contribution of the othermajor unfolding process (involving the 56-69region) has been reduced by stabilisingmutations.

The most spectacular example of adesigned stabilising mutation in the 56-69region in TLP-ste was an engineered disulphidebridge between residues 8 and 60 whichincreased the T50 by as much as 16.7 degrees(82). This result contrasts with the rathermarginal stability effects that were obtainedupon introducing a variety of designeddisulphide bridges in the broad-specificityprotease subtilisin (83). Eijsink et al. (76)propose that the lack of success of theengineered disulphide bridges in subtilisin is atleast partly due to the fact that these bridgeswere introduced in to regions of the proteasethat do not play a dominant role in stability-determining local unfolding processes.

Combination of the five stabilising TLP-ste�thermolysin mutations (A4T, T56A, G58A,T63F, A69P) with the designed mutations S65P,G8C, and N60C yielded one of the most stableproteins ever obtained by protein engineering(84, 85). This 8-fold variant had a half-life of170 minutes at 100 oC and was namedboilysin™. Boilysin was stable for at least 24hours at 90 degrees, a temperature at which thehalf-life of the original enzyme (wild-type TLP-ste) was approximately one minute. It wasshown that, in contrast to wild-type TLP-ste,

boilysin tolerates considerable amounts of urea,guanidinium-HCl and SDS. For example, theenzyme retained approximately 60 percent of itsactivity in the presence of 5 M urea and 40 % ofits activity in the presence of 1 % (wt/vol) SDS(84).

1.8 Engineering hinge bending inthermolysin-like proteases.

Many proteins undergo hinge-bendingmotions during catalysis (86-90). Hinge-bending is the motion of two domains of anenzyme around a hinge point or hinge axis.Through this hinge movement one domain of anenzyme can close onto another to isolate asubstrate from the environment. In several casesglycine residues have been shown crucial forproviding a protein with the necessary (hinge-bending) flexibility (54, 89, 90). Van Aalten etal. (91) showed that mutation of a glycineresidue in the proposed hinge region in retinolbinding protein indeed dramatically reducedretinol binding.

Originally, the crystal structure ofthermolysin was supposed to be that of the pureenzyme (92, 93). When the crystal structure ofB. cereus TLP (TLP-cer) became available, itwas noticed that the active site cleft of thisenzyme was more open than that in thermolysin,resulting from a hinge-bending between the N-and C-terminal domains (54, 86). Puzzled bythis observation, Holland et al. (86) re-examined the original crystallographic data forthermolysin and TLP-cer. They concluded thatthe structure of thermolysin was not that of thefree enzyme, but of the enzyme containing adipeptide in the active site. This peptide was notpresent in the TLP-cer structure. Theseobservations suggested that a hinge-bendingmotion is part of the catalytic mechanism ofTLPs, and that substrate binding yields a closureof the active site (86). Interestingly, a hinge-bending motion quite similar to the oneproposed by Holland et al. was observed when

Chapter 1

14

concerted motions in thermolysin were studiedusing so-called 'essential dynamics' analysis ofmolecular dynamics simulations of thermolysin(89). Originally, Stark et al. (54) suggested thatthe hinge-bending region in TLP-cer andthermolysin is located around glycines 135 and136. This is an attractive proposal, since itplaces the hinge at the beginning of the α-helixconnecting the two domains between whichhinge-bending occurs. In later studies, Hollandet al. proposed a more complex scheme, inwhich bending of the N-terminal α-helix,around Gly78, plays a prominent role. On thebasis of their molecular dynamics simulations,Van Aalten et al. suggested the hinge region inthermolysin to be at both ends of the α-helixconnecting the N- and C-terminal domain(residues 135 and 136 are on the N-terminal endof the connecting α-helix).

The alignment of several TLPs [see (54)or http://www.merops.co.uk/merops/famcards/m4.htm] shows that Gly78 and several glycinesin the α-helix connecting the two domains areconserved in certain groups of TLPs. Residue135 is the most conserved glycine; otherglycines are less well conserved, but acorrelation exists between the disappearance of

certain glycines and the appearance of others.For example, a glycine at 147 is conserved inthose TLPs that have no glycine at position 136.All these glycine residues (78, 135, 136, 141,147, 154) have dihedral angles that arecompatible with some non-glycine amino acidresidues and therefore, are unlikely to representresidues exclusively required for the 3Dstructure of the enzyme. Rather they may berequired for hinge-bending.

1.9 Engineering substrate specificity andactivity.

Rational design of substrate specificity isone of the main goals of protein engineering.Exploitation of enzymes in industry would befacilitated by the ability to rationally modify thesubstrate specificity of an enzyme.Consequently, an extensive body of literatureexists on engineering the substrate specificity ofproteases. The subsite and substratenomenclature regarding specificity issummarized in Fig. 1.7 (94). Three examples ofspecificity determinants are discussed below.

The first and most common example isthe engineering of the substrate binding pocketsto change the substrate specificity. Mei et al.

Figure 1.7. Subsite and substrate nomenclature of proteases. The amino acids of the substrate are countedfrom the cleavage site toward the N-terminus as P1, P2 etc, and towards the C-terminus as P1', P2' etc. Thecorresponding binding pockets on the enzyme are called S1, S2 and S1' and S2'.

Introduction

15

(95) replaced glycines in the S1 subsite ofsubtilisin YaB by larger residues such as alanineand valine. This resulted in an increase inactivity towards substrates with a P1 Ala and asharp decrease in activity towards substrateswith a P1 Phe or Leu. Many other examplesexist in which the preference for largehydrophobic substrates was diminished byreducing the substrate binding pocket sizethrough the replacement of small binding pocketresidues by larger residues (95-97) or byblocking the entrance of the binding pocket(98).

The second example concerns theconversion of trypsin to chymotrypsin. One ofthe most thoroughly studied and now bestunderstood systems is the conversion of trypsinto chymotrypsin and the structural basis ofsubstrate specificity in the serine proteases (99-101). Trypsin (EC 3.4.21.4) and chymotrypsin(EC 3.4.21.1) both belong to the S1 peptidasefamily and catalyze peptide bond cleavage byidentical mechanisms. A serine residue acts as anucleophile and the catalytic residues are in theorder His, Asp, Ser in the primary sequence.Both enzymes are endopeptidases and possessvery similar tertiary structures consisting of twojuxtaposed six stranded β-barrel domains (102,103). The substrate specificity of trypsin,expressed in relative kcat/Km values, is nearly106-fold higher for P1 Arg or Lys containingsubstrates compared to the activity towardsanalogous P1 Phe containing substrates.Conversely, chymotrypsin favours peptidesubstrates possessing Trp, Tyr and Phe at the P1

position, with an overall specificity relative toP1 Lys substrates of up to 104-fold.

Since the structures of the S1 subsites ofthe two enzymes are very similar, the differencein substrate specificity was thought to be asimple property of the local electrostaticenvironment. However, replacement of theprimary binding determinant Asp189 of trypsinwith the analogous Ser189 of chymotrypsin

failed to convert the specificity but, instead,resulted in a poorly performing nonspecificprotease (104). Conversion of trypsin to achymotrypsin-like protease required thesubstitution of four residues in the S1 subsitetogether with the exchange of two adjacentsurface loops, which do not directly contact thesubstrate (105). Inspection of the crystalstructures of the wild-type trypsin andchymotrypsin and those of several mutants,revealed the specificity determinants involved(99). The conserved Gly216, which contacts theP3 residue in both trypsin and chymotrypsin,turned out to be crucial for correct positioningof the substrate in the active site. The differentstructures of the surface loops in trypsin andchymotrypsin maintain Gly216 in distinctconformations, enabling this residue to functionas a specificity determinant despite beingconserved in both proteases.

The study of the trypsin-chymotrypsinsystem has led to a definition of two types ofspecificity determinants (99); primaryspecificity determinants encompassing aminoacids that directly contact the substrate, andsecondary specificity determinants which aremore distantly located elements in the protein.The secondary determinants can act throughvarious mechanisms such as influencing theconformation of primary determinants, as in thecase of Gly216 in trypsin and chymotrypsin, orby modulating the degree of flexibility in thesubstrate binding site. Examples of the latter canbe found in elastase (106, 107) and coenzyme Atransferase (108). The existence of secondaryspecificity determinants imply that substratespecificity is not necessarily determined by alimited set of amino acids in the substratebinding pockets. Instead, substrate specificitycan be a globally distributed propertydetermined by a large part of the protein fold.

The third example is another example ofa specificity determinant which is not located ina subsite. This example relates to the S10 family

Chapter 1

16

of serine carboxypeptidases. Carboxypeptidases(CPD's) catalyze the removal of amino acidsfrom the C-terminus of peptide substrates. TheS10 family of serine carboxypeptidases is agroup of eukaryotic proteases that, based ontheir primary structures, can be divided intothree groups (109), namely those that have asimilar S1 pocket environment as CPD-C, thosethat have a similar S1 pocket as CPD-D and asmall group of unassignable proteases. AllCPD-D like proteases preferentially hydrolyzesubstrates with a P1 Lys as compared toanalogous Leu containing substrates. Amongthe CDP-C carboxypeptidases some areselective for P1 Leu, others for P1 Lys.

Unexpectedly, the comparison ofprimary structures showed that the substratebinding pocket itself is fully conserved in allS10 family members, offering no explanationfor the differences in substrate specificity.However three residues around the S1 pocketwere not conserved. Mutation of these residuesand analysis of their effects showed that thepreference for a P1 Lys originated from the

accessibility of the P1 side chain in the pocket towater, not from a direct interaction between theprotein and the P1 side chain of the substrate(109-112).

The examples referred to above illustratethat the substrate binding pockets play animportant role in determining the substratespecificty. However, other residues outside thebinding pockets can influence the substratespecificity as well.

1.10 Binding modes and geometric effects inhydrophobic subsites.

A number of studies have shown thathydrophobic binding pockets can displaycomplex substrate binding behaviour (113).Different amino acids can show differentbinding modes in which substrates interact withdifferent residues in a hydrophobic bindingpocket. Furthermore, examples exist in whichneighbouring amino acids in the substrateinfluence the exact conformation of a substrateamino acid in a binding pocket. This isillustrated by the following two examples.

Table I.I. Dependence on the fluorogenic group of the substrate of the activity of thermolysin.

kcat/Km P2' SpecificityX =

Fa-GlyaX =

Cbz-GlybX =

Aaf cX =

Fa-GlyaX =

Cbz-GlybX =Aaf c

s-1 ⋅ M-1 × 10-3 ratio kcat/Km

X-Leu-NH2 22 5.1 0.0054 Gly/NH2 3.8 1.2 3.7X-Leu-Gly-OH 83 6.1 0.020 Phe/Gly 3.6 8.2 5.0X-Leu-Phe-OH 300 50 0.10 Ala/Phe 2.9 1.6 4.7X-Leu-Ala-OH 870 78 0.47 Leu/Ala n.d. 1.8 1.2X-Leu-Leu-OH n.d. 144 0.58 Ala/NH2 39.5 15.3 87

Leu/NH2 n.d. 28 107

adata from (116), bdata from (115), cdata from (64). Fa = 3-(2-Furyl)acryloyl, Cbz = benzyloxycarbonyl,Aaf = N-4-methoxyphenylazoformyl, n.d. = not determined.

Chapter 1

16

of serine carboxypeptidases. Carboxypeptidases(CPD's) catalyze the removal of amino acidsfrom the C-terminus of peptide substrates. TheS10 family of serine carboxypeptidases is agroup of eukaryotic proteases that, based ontheir primary structures, can be divided intothree groups (109), namely those that have asimilar S1 pocket environment as CPD-C, thosethat have a similar S1 pocket as CPD-D and asmall group of unassignable proteases. AllCPD-D like proteases preferentially hydrolyzesubstrates with a P1 Lys as compared toanalogous Leu containing substrates. Amongthe CDP-C carboxypeptidases some areselective for P1 Leu, others for P1 Lys.

Unexpectedly, the comparison ofprimary structures showed that the substratebinding pocket itself is fully conserved in allS10 family members, offering no explanationfor the differences in substrate specificity.However three residues around the S1 pocketwere not conserved. Mutation of these residuesand analysis of their effects showed that thepreference for a P1 Lys originated from the

accessibility of the P1 side chain in the pocket towater, not from a direct interaction between theprotein and the P1 side chain of the substrate(109-112).

The examples referred to above illustratethat the substrate binding pockets play animportant role in determining the substratespecificty. However, other residues outside thebinding pockets can influence the substratespecificity as well.

1.10 Binding modes and geometric effects inhydrophobic subsites.

A number of studies have shown thathydrophobic binding pockets can displaycomplex substrate binding behaviour (113).Different amino acids can show differentbinding modes in which substrates interact withdifferent residues in a hydrophobic bindingpocket. Furthermore, examples exist in whichneighbouring amino acids in the substrateinfluence the exact conformation of a substrateamino acid in a binding pocket. This isillustrated by the following two examples.

Table I.I. Dependence on the fluorogenic group of the substrate of the activity of thermolysin.

kcat/Km P2' SpecificityX =

Fa-GlyaX =

Cbz-GlybX =

Aaf cX =

Fa-GlyaX =

Cbz-GlybX =Aaf c

s-1 ⋅ M-1 × 10-3 ratio kcat/Km

X-Leu-NH2 22 5.1 0.0054 Gly/NH2 3.8 1.2 3.7X-Leu-Gly-OH 83 6.1 0.020 Phe/Gly 3.6 8.2 5.0X-Leu-Phe-OH 300 50 0.10 Ala/Phe 2.9 1.6 4.7X-Leu-Ala-OH 870 78 0.47 Leu/Ala n.d. 1.8 1.2X-Leu-Leu-OH n.d. 144 0.58 Ala/NH2 39.5 15.3 87

Leu/NH2 n.d. 28 107

adata from (116), bdata from (115), cdata from (64). Fa = 3-(2-Furyl)acryloyl, Cbz = benzyloxycarbonyl,Aaf = N-4-methoxyphenylazoformyl, n.d. = not determined.

Introduction

17

The first of these examples concernssubtilisin Lentus, also known as savinase, whichis a close relative to subtilisin BPN' (EC3.4.21.62) and belongs to the peptidase familyS8. Investigations into the origin of thespecificity of this subtilisin for substrates withlarge hydrophobic P4 residues revealed that notall substrates interact with the same pocketresidues (113). The orientation of a P4 Leudiffers from that of a P4 Phe, such that theyinteract with different residues of the S4 pocket.The differences in interaction enabled theselective increase in specificity towards eitherP4 Leu or P4 Phe by selectively mutating pocketresidues without changing the activity towardsother substrates (98, 111, 113).

The second example concerns to peptidesubstrates labeled with a fluorogenic groupwhich are widely used to determine thesubstrate specificity of a protease. An oftenignored problem using such peptides is the factthat the fluorogenic group can have aconsiderable effect on substrate preference. Theeffects of different fluorogenic groups becameapparent when the substrate specificity of TLNwas determined with Morihara-Tsuzuki peptidesubstrates that only differed in their fluorogenicgroup (114). Table I.I clearly demonstrates thiseffect.

The Morihara-Tsuzuki peptide series is aset of five peptides commonly used to determinethe P2' preference of TLPs (115). The data inTable I.I show that the specificity for thesepeptides strongly depends on the nature of thefluorogenic group. This range of peptides with avariable P2' residue has been used with threedifferent fluorogenic groups, indicated in thetable. Using Cbz as the fluorogenic group theactivity is increased only 28-fold when the P2'NH2 was changed to P2' Leu, which is in sharpcontrast with a 107-fold increase in activitywhen Aaf was used as the fluorogenic group.The effect of the fluorogenic group probablyoriginates from the fact that the group itself

occupies a binding pocket, which in TLPs isusually either the S2 or S1 subsite. The bindingof an amino acid or fluorogenic group in asubsite can influence the binding mode of therest of the substrate resulting in a change inactivity.

The examples referred to above illustratethat the configuration of a substrate is importantfor the activity of the enzyme. The examplesalso illustrate that the conformation of aparticular side chain of the substrate maydepend on the other amino acids of thesubstrate. Thus, if peptide substrate data areused to discuss any mechanism or change inspecificity of a protease, these effects should betaken into account.

1.11 Chemical modification and substratespecificity.

Prior to the development of site-directedmutagenesis techniques only chemical methodswere available to protein chemists to alterenzyme properties (116). One of the mainproblems of chemical modification of enzymesis that the extent and precise location of themodifications often remain uncertain becausemost reagents are unspecific. Furthermore,heterogeneous mixtures are often produced.Despite such disadvantages, combiningchemical modification and site directedmutagenesis provide a unique handle for proteinmodification, because chemical modification ofthe substituted amino acid offers the possibilityto introduce virtually any desired molecule at aspecific site in the protein. This approach allowsone to introduce unnatural amino acid sidechains and to circumvent the limitations instructural variations imposed by the occurrenceof only 20 natural amino acids.

An example of the combinedmutagenesis and chemical modificationapproach to modify the substrate specificity ofsubtilisin concerns the introduction of a uniquecysteine in the S1 binding pocket, followed by

Chapter 1

18

its chemical modification withmethanethiosulfonate reagents to generatechemically modified mutant enzymes (116-118). A potential problem with this approach isthe size reduction of the binding pocket due tothe introduced chemical modification. Studieswith subtilisin indeed showed some sizeexclusion effects (118) although a proper choiceof the modification site could avoid some ofthese problems. The various available reagentsoffer the possibility to introduce novelfunctionalities in a binding pocket, such asmultiple negative charges. In this wayencouraging results concerning the alteration ofsubstrate specificity have already been obtained(119).

Another possible application of chemicalmodification of proteins is the production ofglycosylated heterologous proteins byprokaryotes. A persistent problem of eukaryoticgene expression in prokaryotes is the lack ofglycosylation of the expressed proteins. Regio-selective glycosylation of subtilisin wasobtained through site directed mutagenesis andsubsequent chemical modification afterpurification of the protein (120, 121). This is animportant advance for the production ofproperly glycosylated eukaryotic proteins inprokaryotes.

1.12 Scope of this thesis.This thesis describes the engineering of

the activity and substrate specificity of severalthermolysin-like proteases from Bacillus. Themajority of the experiments were performedwith the thermolysin-like protease from Bacillusstearothermophilus (TLP-ste) and thermolysinfrom Bacillus thermoproteolyticus (TLN).

After a general introduction in chapter 1,chapter 2 discribes the characterization of TLPswith peptide substrates, as well as HPLCanalysis of β-casein digests. The results indicatethat the M4 family is a homogeneous family interms of catalysis, even though there is a

significant degree of amino acid sequencevariation. The results of this study show thatdifferences in substrate specificity within theM4 family do not correlate with overallsequence differences but depend on a smallnumber of identifiable amino acids. Indeed,molecular modeling, followed by site directedmutagenesis of one of the substrate bindingpocket residues of the TLP of B.stearothermophilus, converted the catalyticcharacteristics of this variant into that ofthermolysin.

Chapter 3 shows the importance ofconserved glycines in the proposed hinge-bending regions by analyzing the effects ofGly�Ala mutations on catalytic activity.Comparisons of effects on kcat/Km for varioussubstrates with effects on the Ki forphosphoramidon, suggested that the mutation atposition 78 primarily had an effect on substratebinding, whereas the mutations at positions 135and 136 primarily influence kcat. The apparentimportance of conserved glycine residues inproposed hinge-bending regions for TLPactivity supports the idea that hinge-bending isan essential part of catalysis.

Chapter 4 discribes the properties of themajor specificity determining hydrophobic S1'pocket. The results indicate that the S1' Phe/Leupreference can be changed by increasing theactivity towards substrates with a P1' Phe. Inaddition, the results obtained with TLN andTLP-ste support the quality of the TLP-stemodel and indicate that the substrate preferenceof all TLPs can be modified in a similar manneras the substrate specificity of TLP-ste. The 16-fold increase in activity of the Leu202Tyrmutant towards a P1' Phe containing substrate isone of the highest found in the literature for asingle mutant.

Chapter 5 examines the possibility ofchanging the active site electrostatics of thethermolysin-like protease from B.stearothermophilus by inserting or removing

Introduction

19

charges on the protein surface by site-directedmutagenesis. The results show that the effectson the kcat/Km of single point mutations are non-additive, even in cases where the pointmutations are 10Å or more removed from theactive site Zn2+ and separated from each otherby up to 25Å. This suggests that electrostaticnetworks are probably more complex thanpreviously thought and that possible othereffects, such as active site dynamics, play animportant role in determining the active site

electrostatics. Several mutations caused asignificant increase in enzyme activity, the mostactive mutant being almost four times as activeas the wild-type. The shape of the pH-activityprofile was changed significantly. Remarkably,this was achieved without large changes of thepH-optimum of the enzyme.

Chapter 6 contains the summary and ageneral discussion of the results and conclusionsof the present work.

M4 family specificity determinants

21

Substrate specificity in the highly heterogeneousM4 peptidase family is determined by a small

subset of amino acids.

AbstractThe members of the M4 peptidase

family are involved in processes as diverse aspathogenicity and industrial applications. Forthe first time a number of M4 family members,also known as thermolysin-like proteases(TLPs), has been characterized with an identicalsubstrate set and a uniform set of assayconditions. Characterization with peptidesubstrates, as well as HPLC analysis of β-caseindigests, shows that the M4 family is ahomogeneous family in terms of catalysis, eventhough there is a significant degree of aminoacid sequence variation. The results of thisstudy show that differences in substratespecificity within the M4 family do not correlatewith overall sequence differences but depend ona small number of identifiable amino acids.Indeed, molecular modeling, followed by sitedirected mutagenesis of one of the substratebinding pocket residues of the TLP of Bacillusstearothermophilus, converted the catalyticcharacteristics of this variant into that ofthermolysin.

IntroductionThermolysin-like proteases (TLPs) are

members of the peptidase family M4 (122) ofwhich thermolysin (TLN; EC 3.4.24.27) is theprototype. The phylogenetic tree for the M4family is shown in Fig. 2.1A. The familycontains only secreted eubacterialendopeptidases, from both Gram-positive andGram-negative sources. All members of thiscomprehensive family are produced as pre-pro-proteins. During export the pre-sequence(signal sequence) is cleaved off, whereas theprosequence has been shown to assist in proper

folding, by acting as a molecular chaperone(123). In addition, it has been shown that theprosequence can act as a specific inhibitor(124), thus preventing (125) unwantedproteolytic activity in the cytoplasm (123). Themature enzymes are all of moderate size, around35 kDa (316 amino acids for thermolysin).These proteases contain the typical HEXXHamino acid motif, require Zn2+ ions for theiractivity and contain multiple Ca2+ ions (up tofour) for stability. All enzymes are optimallyactive at neutral pH (122, 126).

For several of these enzymes three-dimensional structures are available (52, 54, 56,127). In fact, thermolysin was among the firstproteins for which the structure was solved.Although considerable sequence diversity existswithin this family (Fig. 2.1B), there is a highdegree of structural conservation. All membersfor which the structure has been solved, wereshown to consist of two major domains. The N-terminal domain contains mainly β-sheets,whereas the C-terminal domain predominantlycontains α-helices. The active site is located inthe cleft between these two domains. In thoseenzymes of which the structure has beendetermined, the catalytically essential Zn2+ ionis located at the bottom of this cleft (Fig. 2.2).In a significant number of published structuresin which TLN was co-crystallized withinhibitors (52, 57-61), the residues involved incatalysis could be identified.

The family also includes enzymes frompathogens such as Legionella, Listeria,Clostridium, Staphylococcus, Pseudomonas andVibrio. For example, pseudolysin, the TLPfrom Pseudomonas aeruginosa, has been shownto cause tissue damage by degrading collagens,

Chapter 2

22

1 M04.015 Bacillus thuringiensis TLP (-)2 M04.015 Bacillus cereus TLP (P05806)3 M04.015 Lactobacillus sp. TLP (Q48857)4 M04.015 Bacillus megaterium TLP (Q00891)5 M04.001 Bacillus caldolyticus thermolysin (P23384)6 M04.001 Bacillus sp. thermolysin (Q59223)7 M04.001 Alicyclobacillus acidocaldarius thermolysin (Q43880)8 M04.001 Bacillus stearothermophilus NprT protein (P06874)9 M04.001 Bacillus stearothermophilus NprS protein (P43133)10 M04.001 Bacillus thermoproteolyticus thermolysin (P00800)11 Brevibacillus brevis unassigned peptidase (M4) (P43263)12 Paenibacillus polymyxa unassigned peptidase (M4) (P29148)13 M04.012 Bacillus subtilis neutral protease B (P39899)14 Clostridium histolyticum unassigned peptidase (M4) (-)15 M04.011 Clostridium perfringens lambda toxin (Q46237)16 M04.009 Staphylococcus chromogenes aureolysin (AAF32312)17 M04.009 Staphylococcus epidermidis aureolysin (P43148)18 M04.014 Bacillus amyloliquefaciens bacillolysin (P06832)19 M04.014 Bacillus subtilis bacillolysin (P06142)20 M04.008 Listeria monocytogenes TLP (Listeria) (2) (P34025)21 M04.008 Listeria monocytogenes TLP (Listeria) (1) (P23224)

FigThseqphyfro/mC. thi

A

B

C

22 Streptomyces coelicolor unassigned peptidase (CAB66423)23 Erwinia carotovora unassigned peptidase (M4) (Q99132)24 M04.007 Enterococcus faecalis coccolysin (Q47786)25 Streptomyces coelicolor SC3D11.04C protein (CAB76001)26 Streptomyces griseus metallopeptidase II (-)27 Renibacterium salmoninarum unassigned peptidase (M4) (hemolysin) (P55111)28 M04.003 Vibrio anguillarum vibriolysin (P43147)29 M04.003 Vibrio proteolyticus vibriolysin (Q00971)30 M04.003 Vibrio vulnificus vibriolysin (O06694)31 M04.003 Vibrio cholerae vibriolysin (P24153)32 M04.016 Aeromonas hydrophila pro-aminopeptidase processing protease (-)33 M04.016 Aeromonas caviae pro-aminopeptidase processing protease (Q9R9S7)34 M04.005 Pseudomonas aeruginosa pseudolysin (P14756)35 M04.006 Legionella longbeachae vibriolysin hom. (P55110)36 M04.006 Legionella pneumophila vibriolysin hom. (P21347)37 Bacillus cereus unassigned peptidase (NprB protein) (CAB69809)38 Serratia marcescens unassigned peptidase (M4) (Q06517)

ure 2.1. A. Phylogenetic tree of peptidase family M4.e 5 peptidases used in this study are indicated. B. Key touences with Swiss Prot and MEROPS identifiers. Thelogenetic tree and key to the sequences were adapted

m the MEROPS database (http://www.merops.co.ukerops/famcards/m4.htm) with permission of the authors.Sequence identity matrix of the 5 peptidases used ins study.

M4 family specificity determinants

23

elastin and fibronectin (128), whereas the TLPsfrom Listeria spp. appear to be involved in thematuration of specific virulence factors (129).Furthermore, the active site organization of M4peptidases exhibits similarity to those of anumber of eukaryotic metallopeptidases, inparticular to members of the matrixmetalloproteases (MMP’s)(130). These latterenzymes were shown to be involved in anumber of important processes in man,including the processing of precursors that playmodulation roles in the formation of tumors. Inaddition, metallo-endopeptidases are involved inmany cellular processes such as exocytosis, cell-cell fusion and neuropeptide hydrolysis (131).Consequently, metalloproteases of the M4family have attracted increasing attention asmodel proteins for the development of specificinhibitors that can be applied for diseasetreatment (132). In addition, several members ofthis protease family are applied in industry, e.g.in baking, brewing and leather processing (24).Thermolysin is being used for the synthesis ofthe artificial sweetener aspartame (24).

In this study we have characterizedseveral thermolysin-like proteases (TLPs) ofBacillus and Staphylococcus species. Theavailability of an impressive amount ofsequence, structural and kinetic data renders thisgroup of proteases an ideal subject for rationaldesign strategies. Although some of the familymembers have been characterized individually(114, 115, 126, 133, 134) a consistentcomparison with an identical substrate set and auniform set of assay conditions has never beenconducted. Previously it was suggested thatTLPs exhibit a preference for large hydrophobicP1' residues (Leu or Phe) (114, 122, 130, 133).In addition, it has been demonstrated that the S1'pocket is the major determinant of the substratespecificity (114). Here we show that thethermolysin-like protease (TLP) family is anextremely homogeneous family in terms ofcatalysis, even though there is a significant

degree of sequence variation. Furthermore, weshow that existing differences in specificity andactivity between two individual members can becanceled by a single amino acid substitution.

Materials and MethodsGenetics.

The nprM gene encoding thermolysin(TLN) of B. thermoproteolyticus (135), the nprTgene encoding the TLP of B.stearothermophilus CU21 (136)(TLP-ste), thenprC gene encoding the TLP of B.cereus(124)(TLP-cer) and the nprB gene encoding theTLP of B.subtilis (137)(TLP-sub) were cloned,subcloned, and expressed as describedpreviously (80). The purified TLP ofStaphylococcus aureus (138)(Aureolysin orTLP-sau, EC 3.4.24.29) was kindly provided byDr. J. Potempa. Site-directed mutagenesis wasperformed by the PCR-based mega-primermethod, essentially as described by Sarkar andSommer (139). Mutagenic primers weredesigned such that mutant clones could berecognized by the appearance or disappearanceof an endonuclease restriction site (80). Thenucleotide sequences of mutated fragments ofthe nprT gene were verified by DNA sequenceanalysis.

Modeling and mutant design.A three-dimensional model of TLP-ste

was built on the basis of homology withthermolysin (86% sequence identity), using themolecular modeling program WHAT-IF (140).The modeling procedures have been describedin detail elsewhere (78). Because of the highsequence similarity the model was expected tobe sufficiently reliable for prediction andanalysis of the effects of most amino acidsubstitutions (78, 141). This has beenconfirmed by the fact that the model has beenused for the successful design of variousstabilizing mutations (43, 82, 142, 143).Throughout this paper, residues in all TLPs are

Chapter 2

24

numbered according to the numbering ofcorresponding residues in thermolysin.

Production and Characterization of Enzymes.Production and purification of the

enzymes were performed as described earlier(80, 144). Before determining the kineticparameters, protease preparations were desaltedto 20 mM NaAc pH 5.3, 5 mM CaCl2 and 20%isopropanol using pre-packed PD-10 gelfiltration columns supplied by AmershamPharmacia.

Specific activities of the TLPs towardscasein were determined according to a methodadapted from Fujii et al. (136): approximately0.5 µg of protease was incubated in 1 ml of 50mM 2-amino-2-(hydroxymethyl)-1,3-propane-diol (Tris⋅HCl) (pH 7.5) containing 0.8%(wt/vol) casein and 5 mM CaCl2 at 37 °C for 1h. The reaction was quenched by the additionof 1 ml of a solution containing 100 mM tri-chloro-acetic acid (TCA), pH 3.5. One unit ofactivity is defined as the amount of enzymeactivity needed to liberate a quantity of acid-soluble peptide corresponding to an increase inA275nm of 0.001 per min.

The kcat/Km and Km values forfurylacryloylated di- and tripeptides of theenzymes were determined at 37 °C in athermostated Perkin-Elmer Lambda 11spectrophotometer. The reaction mixture (1 ml)contained 50 mM Tris, 50 mM 4-morpholineethanesulfonic acid (MES) (pH 7.0),5 mM CaCl2, 5% DMSO, 0.5% 2-propanol,0.01% Triton X-100 and 100 µM to 2.5 mM ofsubstrate, and the reaction was followed bymeasuring the decrease in absorption at 345 nm(∆ε345 = -317 M-1⋅cm-1 )(114). All substrateswere supplied by Bachem. Stock solutions ofthe furylacryloylated dipeptides 3-(2-furylacryloyl)-L-glycyl-L-leucine-amide(FaGLa) and 3-(2-furylacryloyl)-L-glycyl-L-phenyl-amide (FaGFa), and of the

furylacryloylated tripeptides 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine(FaGLA) and 3-(2-furylacryloyl)-L-glycyl-L-phenylalanine-L-leucine (FaGFL) wereprepared by dissolving the peptides in Me2SO.The apparent second order rate constant kcat/Km

was determined by varying the enzymeconcentrations (over a 50-fold range) underpseudo-first-order conditions and measuring theinitial activity, essentially according to themethod described by Feder (114).

The Ki for N-[α-L-rhamnopyranosyl-oxyhydroxyphosphinyl]-L-leucine-L-tryptophan(phosphoramidon) was determined by a 10 minpreincubation of a 0.1 nM protease solutionwith varying concentrations of the inhibitor (10-

8 to 10-3 M), in 50 mM Tris, 50 mM MES (pH7.0), 5 mM CaCl2 , 0.01% Triton X100.Subsequently, enzyme activity was determinedusing 100 µM FaGLA as substrate. Ki ’s werecalculated by the method described by Hunterand Downs (145).

For the determination of thermalstability 0.1 µM purified protease solutions (in20 mM sodium acetate, pH 5.3, 5 mM CaCl2,0.01% Triton X-100, 0.5% 2-propanol, and 62.5mM NaCl) were incubated at varioustemperatures for 30 min, after which theresidual proteolytic activity was determinedwith casein as a substrate (136). Thermalstability was quantified by T50, being thetemperature giving 50% residual activity after a30 min period of incubation (78, 81).

The proteolytic properties of the mutantenzymes towards β-casein (Sigma-Aldrich),were determined by means of HPLC. β -casein(1 mg ml-1) was incubated in 50 mM Tris, 50mM MES (pH 7.0), 5 mM CaCl2, 0.01% TritonX-100 with each of the TLP variants at a molarratio of 1,000:1 at 37 °C for 24 hrs. Thepeptides resulting from hydrolysis werederivatized with dansyl-chloride. Theproteolytic products were separated by loading a

M4 family specificity determinants

25

sample corresponding to 50 µg β-casein on areversed phase column (RP-304, Bio-RadLaboratories). The mobile phase used was 50mM NaAc, pH 5.2. Peptides were eluted with alinear gradient of 0-60% acetonitrile in 30 minat a flow rate of 1 ml min-1. Absorption of theeluting peptides was monitored at 254 nm.

ResultsEnzymatic properties towards casein.

To investigate the activity of the variousM4 proteases on large protein substrates, theactivity towards casein was determined. Caseinwas selected as a standard substrate for activitymeasurements because it behaves as anoncompact and largely flexible structure (146),thus rendering all scissionable motifs accessibleto the same extent for the various proteases atall temperatures employed. Indeed, we haveshown previously that digestion of β-caseinwith TLP-ste at different temperatures yieldedidentical degradation products (84). The resultsare shown in Table II.I. Most of the wild-typeenzymes show similar specific activities, with avariation of a factor of approximately 3. Themajor exception is TLP-cer, which is much lessactive on casein than the other enzymes tested.

To determine the thermal stabilityand the optimal temperature for catalysisof the various proteases, we determinedthe T50 (79) values and the temperaturedependence of activity towards casein.The T50 values are given in Table II.I.These values correlate well with thetemperature optima of the TLPs as shownin Fig. 2.3, in the sense that the mostthermally labile protease shows the lowestoptimum temperature. To facilitatecomparison, the maximum activity of thedifferent TLPs has been normalized to100%.

Between closely related TLPs acorrelation exists between the degree of

sequence identity and the difference in thermalstability (see Table II.I for a comparison of thesequence identity and the ∆T50 values). In allcases, the temperature optimum is just belowthe T50 value determined, which is a direct resultof the experimental procedures: the T50 valueswere determined with a 30 min incubationperiod followed by determination of theremaining activity at 37 °C, whereas thetemperature optima were determined during a 1hr incubation period at the indicatedtemperatures. As a consequence, this longerincubation period can be expected to lead to ahigher degree of inactivation at the elevatedtemperatures.

Inspection of Fig. 2.3 shows that theshape of the curve of TLN differs as comparedto those of the other TLPs. Of the enzymestested only TLP-sub and TLN shows Arrheniusbehavior: the activity increases exponentiallywith the temperature. TLP-sau deviates fromthe other TLP’s by showing an unexpectedlybroad temperature optimum, suggesting thatthermal (in)activation of this protease mightdiffer from that in the other proteases.

Table II.I Specific activity and thermostability of TLP’s.

SpecificActivitya

T50b ∆T50

to TLNSequence identityto TLN

Units × 10-3 °C °C °C

TLP-cer 1.4 68.6 18.3 73TLP-ste 29 73.4 13.5 86TLN 42 86.9 0.0 100TLP-sub 47 58.6 28.3 48TLP-sau 88 55.1 31.8 45

TLP-ste F133L

57 74.9 12.0 86

aStandard deviations are less than 10 % of the values given.bThe error margin amounts to approximately 0.3 ºC.

Chapter 2

2

Ct

spwaItPTrsT

shown by the Phe/Leu ratio for dipeptides. Incontrast, the M4 family is often described ashaving an equal P1' preference for Leu and Phe(114, 122, 130, 133). The diversity or similarityin primary amino acid sequence (Fig 2.1B), is

Fsthαt(

igure 2.2 Ribbons diagram of thermolysin. The leftide shows the predominantly β-sheet containing N-erminal domain; the right side the predominantly α-elical C-terminal domain. The centre shows the central-helix which is the bottom of the active site cleft with

he catalytic Zn2+ ion (large sphere). Four Ca2+ ionssmall spheres), involved in stability, are also shown.

6

atalytic properties of TLPs on di- andripeptide substrates.

To determine the P1' substratepecificity, the activities of the various M4roteases towards di- and tripeptide substratesere determined. Their activities on dipeptide

nd tripeptide substrates are shown in TablesI.II and II.III, respectively. The results showhat both substrates with a Leu as well as with ahe as P1' residue are efficiently hydrolyzed byLPs. As with casein, TLP-cer shows a

elatively low activity towards dipeptideubstrates. With dipeptide substrates, mostLPs prefer Leu over Phe at the P1' position, as

Figure 2.3 Temperature optima of TLPs determinedwith casein as substrate. The maximum activity of eachTLP is set as 100%. ΟTLP-cer, �TLP-ste, TLN,�TLP-sub, ∆TLP-sau, �TLP-ste F133L

M4 family specificity determinants

27

not reflected in either different or similarcleavage efficiencies for the peptide substratestested. In fact, the substrate pecificity of TLN ismuch more similar to that of TLP-sub than tothat of TLP-ste, contrary to what might beexpected on the basis of sequence similarity

(45% and 86% identity, respectively). With theexception of TLP-cer, all activities on peptidesubstrates are less than one order of magnitudedifferent from those of TLN.

In contrast, the inhibition constant forthe inhibitor phosphoramidon, which wasspecifically designed for TLN, seems tocorrelate with the sequence difference. TheTLPs that are phylogenetically close to TLN aremuch more sensitive to phosphoramidon ascompared to those that are more distant.

HPLC characterization of β-casein digests.To examine whether differences in

substrate specificity can be observed on largepeptide substrates, HPLC analyses wereperformed on β-casein hydrolysates by thevarious TLPs. Fig. 2.4 shows a detail of the RP-HPLC analyses of the peptides that were formedupon digestion of β-casein with the TLPs. Anumber of characteristic and reproducibleproducts could be identified for each of theTLPs. Although the preference towards thesmall peptides used showed little variation,differences in the digestion patterns of β-caseinare clearly detectable. This illustrates thatdifferences can be much more readily detectableon large protein substrates than on small peptidesubstrates.

Table II.III Activity of TLPs on tripeptide substrates.

kcat/KM PheFaGLAa FaGFLa Leu

s-1⋅M-1 × 10-5 ratio kcat/KM

TLP-cer 0.17 0.56 3.4TLP-ste 0.56 8.30 14.9TLN 2.05 3.42 1.7TLP-sub 0.79 0.42 0.5TLP-sau 0.18 0.33 1.8

TLP-ste-F133L 1.77 4.34 2.5

aStandard deviations are less than 15 % of the valuesgiven.

Table II.II Activity of TLPs on dipeptide substratesand inhibition constants for phosphoramidon.

kcat/KM Phe Kib

FaGLaa FaGFaa Leu Inhibitionc

s-1⋅M-1 × 10-3 ratio kcat/KM nM

TLP-cer 0.64 0.46 0.7 2.1x102

TLP-ste 2.20 3.40 1.5 13TLN 12.25 3.88 0.3 21TLP-sub 3.20 0.17 0.1 6.1x103

TLP-sau 3.47 5.48 1.6 3.0x103

TLP-ste F133L 17.15 6.31 0.4 17

aStandard deviations are less than 15 % of the valuesgiven. b Phosphoramidon. cStandard deviations are lessthan 10 % of the values given.

Figure 2.4. Detail of HPLC diagrams of ββββ-caseindigests. 1. TLP-ste, 2. TLP-ste F133L, 3. TLN, 4. TLP-sub, 5. TLP-sau, 6. TLP-cer

Chapter 2

28

Site

diffvaridissdiffthe modiffmemandare conlatteextrfeatThearouthatcommofeatdiff

FLs

igure 2.5. Stereo view line drawing of the S1' pocket, showing the P1' Leu side chain, coordinated by theeu202 and the 133 residue. Residue 133 is a Leu in TLN and a Phe in TLP-ste. Mutation Phe133Leu in TLP-

te is predicted to change the S ’ specificity of TLP-ste into that of TLN.

-directed mutagenesis of the active site.The results presented above suggest that

erences in substrate specificity between TLPants are not correlated with overall sequenceimilarities. To examine whether sucherences might be reflected in the structure ofactive site and substrate binding pockets,

lecular modeling of the active sites of theerent variants was employed. For several

bers of the family (TLN, TLP-cer, TLP-sau elastase) high resolution X-ray structuresavailable. In addition, models have been

structed for TLP-sub and TLP-ste. Ther models have previously been shownemely useful for identifying structuralures involved in thermal stability (78, 84). fact that the amino acid conservation in andnd the active sites is very high, suggests

they are structurally similar. We decided topare the active sites of TLN and TLP-ste in

re detail in order to identify structuralures that could explain the observederences in substrate specificity. The two

enzymes are highly similar (86 % sequenceidentity). In particular, in the active site regionsthe sequence conservation is very high.Therefore, the constructed model for TLP-ste isexpected to be highly reliable in this region.

Close inspection of the model of TLP-ste and careful comparison with the TLNstructures available, revealed that one of themajor differences between the two TLPs in theactive site region concerns residue 133, which isa Leu in TLN but a Phe in TLP-ste. The S1'subsite is composed of the side chains ofPhe130, Phe/Leu133, Val139 and Leu202.Furthermore, inspection of the S1' pocket andthe conformation of the various P1' side chainsin TLN-inhibitor complexes (1TLP.PDB, 1-7TMN.PDB, 4-8TLN.PDB) shows that the P1'side chain is sandwiched between the 133 andthe 202 S1' residues (Fig. 2.5).

It might be anticipated that the largePhe133 residue in the S1' pocket will influencethe binding of substrates in this specificity-determining pocket to a considerable extent. Totest this hypothesis, Phe133 in TLP-ste was

1

M4 family specificity determinants

29

substituted by Leu and the effects on substratespecificity were determined. As documented inTables II.II and II.III, the TLP-ste mutant showsenzymatic characteristics on di- and tripeptidesthat are much more TLN-like than TLP-ste-like.In addition, this mutation almost doubled theactivity towards casein (Table II.I).Furthermore, the RP-HPLC patterns obtainedwith the TLP-ste F133L mutant showed someTLN-specific peaks, whereas some TLP-stespecific peak continue to be present as well.However, the temperature optimum, the shapeof the temperature curve and the thermalstability of the single mutant remained identicalto wild-type TLP-ste.

DiscussionThe present study shows a correlation

between the thermal stability and sequenceidentity of the various TLPs. This correlationwith sequence identity does not exist for thedifferences in activity and specificity on bothpeptide substrates and casein. Althoughdifferences in specificity on the peptidesubstrates used are relatively small, HPLCanalysis of digestion patterns of β-casein doesshow specific digestion patterns. The fact thatthe differences in activity and specificity can becanceled by mutating one amino acid in asubstrate binding pocket, indicates that notoverall sequence differences but a small set ofidentifiable amino acid residues is responsiblefor the differences in performance of theseenzymes.

The comparison of the thermostability ofclosely related TLPs, showed that a correlationexists between the sequence identity and thedifference in thermal stability. However,inspection of Fig. 3 shows that TLP-sub andTLN are the only two enzymes of which thethermal activation shows Arrhenius likebehavior. A previously described hyperstablevariant of TLP-ste (84) does not show thisbehavior (147). Thus it seems unlikely that

thermal stability underlies this differencebetween these two and the remainder of theenzymes studied. Rather a process such ashinge-bending (89, 148) could be a more likelycause for the difference between these two setsof enzymes.

The comparison of the enzymaticperformance, i.e. activity and specificity, ofTLPs from Bacilli and Staphylococcus indicatesthat overall divergence in primary sequence isnot correlated with differences in activity andsubstrate specificity. In contrast, local sequencedifferences in the active site and bindingpockets seem to be responsible for the majorityof the differences in activity and substratespecificity. This hypothesis is supported by theobservation that both the kcat/Km for the di- andtripeptide substrates differ less than 1 order ofmagnitude between the various enzymes, withthe exception of TLP-cer, and, as shown by theTLP-ste F133L mutant, the observation that theactivity and substrate specificity of one variantcan be changed into that of another by mutatingjust one binding pocket residue. However, thishypothesis seems to be contradicted by theapparent relation between the Ki forphosphoramidon and the sequence difference.This relation can be explained by the fact thatthe most important residue for phosphoramidonbinding is Phe114 (52, 61, 149) present in bothTLN and TLP-ste (low Ki) whereas the 114position in TLP-cer, -sub and –sau is occupiedby an Ala (high Ki).

The similarity in activity and specificityof the various TLP’s towards peptide substratesdoes not exclude the possibility that overallsequence differences can play a role in activityand specificity towards larger proteinaceoussubstrates. Analysis of the digestion patterns ofβ-casein indicated that there are cleardifferences in substrate specificity on proteinsubstrates. However, the mutant TLP-steF133L, which changes the specificity of TLP-ste to that of TLN on peptide substrates, also

Chapter 2

30

changes the digestion pattern on β-casein into amore TLN like pattern. Although otherexplanations cannot be excluded, this suggeststhat the observed differences in specificity aremainly caused by differences in the active siteand binding pockets and not by overall sequencedifferences.

The present study is the first example ofan approach in which the enzymatic andcatalytic properties of a significant number ofmembers of the M4 peptidase family arecompared under identical conditions. The needof such a comparison is obvious, in view of theroles of members of this family in processes asdiverse as pathogenicity and industrialapplications. The notion that overall differencesin sequence do not correlate with substratespecificity enabled us to modify the substrate

specificity by site-directed mutagenesis of thoseresidues directly involved in substrate bindingand catalysis. Indeed, a single amino acidsubstitution converted catalytic characteristicsof one family member into that of another.Consequently it can be envisaged that specificinhibitors, for example to be used for blockingdisease-related members of the MMP family,can be designed on the basis of amino acidresidues identified in TLPs. Thus, this studyprovides additional arguments for the potentialof TLPs as a model system in the search fornovel MMP inhibitors. Both for thedevelopment of specific inhibitors as well as forthe improvement of biocatalysts, a betterunderstanding of existing relations betweensequence, structure and function is ofconsiderable importance.

Catalytic hinge-bending motions

31

Probing catalytic hinge-bending motions inThermolysin-Like Proteases by Gly ���� Ala

mutations.

AbstractThe active site of thermolysin-like

proteases (TLPs) is located at the bottom of acleft between the N-terminal and C-terminaldomain. Crystallographic studies have shownthat the active-site cleft is more closed inligand-binding TLPs than in ligand-free TLPs.Accordingly, it has been proposed that TLPsundergo a hinge-bending motion duringcatalysis resulting in “closure” and “opening” ofthe active-site cleft. Two hinge regions havebeen proposed. One is located around aconserved glycine 78; the second involvesresidues 135 and 136. The importance ofconserved glycine residues in these hingeregions was studied experimentally byanalyzing the effects of Gly�Ala mutations oncatalytic activity. Eight such mutations weremade in the TLP of Bacillus stearothermophilus(TLP-ste) and their effects on activity towardscasein and various peptide substrates weredetermined. Only the Gly78Ala, Gly136Ala,and Gly135Ala + Gly136Ala mutants decreasedcatalytic activity significantly. These mutantsdisplayed a reduction in kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of73%, 62%, and 96% respectively. Comparisonsof effects on kcat/Km for various substrates witheffects on the Ki for phosphoramidon, suggestedthat the mutation at position 78 primarily had aneffect on substrate binding, whereas themutations at positions 135 and 136 primarilyinfluence kcat. The apparent importance ofconserved glycine residues in proposed hinge-bending regions for TLP activity supports theidea that hinge-bending is an essential part ofcatalysis.

IntroductionThermolysin-like proteases (TLPs) are a

group of homologous metalloendopeptidasesfrom Bacillus with similar enzymaticcharacteristics. The amino acid sequences ofseveral TLPs have been determined [see (122)or the Merops data base (http://www.merops.co.uk/merops/famcards/m4.htm)] and the three-dimensional structures of thermolysin ofBacillus thermoproteolyticus (52), the TLP fromBacillus cereus (53), aureolysin fromStaphylococcus aureus (56) and elastase fromPseudomonas aeruginosa (55) have been solvedby X-ray crystallography. All TLPs have asimilar fold, consisting of an α-helical C-terminal domain and an N-terminal domain thatconsists mainly of β-strands. The domains areconnected by a central α-helix (residues 137-150). This helix is located at the bottom of theactive-site cleft and contains several of thecatalytically important residues [(93); Fig. 3.1].

Holland et al. (86) superposed thecrystal structures of thermolysin, TLP-cer andelastase [the homologous TLP fromPseudomonas aeruginosa; (55)]. They observeda hinge-bending displacement between the N-and C-terminal domains. The hinge-bendingangle between the two domains was larger(meaning a more open active-site cleft) in TLP-cer than in thermolysin. A comparison of the 3Dstructures of elastase crystallized with andwithout inhibitors bound to the active-site (55,86), revealed that the structure of this remoteTLP family member was more closed when aligand is bound. Further refinement of thethermolysin electron density map revealed thatthe active-site contained a dipeptide (valine-

Chapter 3

32

lysibe m

mentherregiglyc(thethe pro

Figure 3.1. Stereo C-αααα trace of thermolysin. Residues 1-135 are shown with dotted lines, and the α-carbons ofGly135 and Gly136 are marked with small spheres. The active site residues that are involved in zinc binding(His142, His146, and Glu166) and catalysis (Glu143, Tyr157, and His231) are added in ball-and-stickrepresentation. The five single spheres are the zinc and the four calciums; the zinc is labeled. The structure givenis that of a closed TLP. The axis describing the hinge-bending displacement is observed by comparing TLPstructures in the vicinity of residues 78, 135, and 136 (see (86) for details). Opening of the active-site cleft isassociated with a change in hinge-bending angle of approximately 6° (86), as well as a small change in thedihedral angles of Gly135 and Gly136 (54).

ne), explaining why thermolysin appeared toore closed than TLP-cer (86, 150).

Stark et al. (54) also noticed the above-tioned difference between TLP-cer andmolysin. These authors proposed the hingeon to reside near two (rather conserved)ine residues at positions 135 and 136rmolysin numbering; Fig. 3.1 and 3.2). Onbasis of inhibitor studies, Thayer et al., (55)posed the presence of a hinge in the

corresponding location in elastase. A similarconclusion was reached by Van Aalten et al.(89), who studied the dynamics of thermolysinby essential dynamics analyses of moleculardynamics simulations (151). On the basis ofdetailed crystallographic studies of variousTLPs, Holland et al. (86, 150) suggested thatalso the conserved glycine 78 could beimportant for hinge-bending (Fig. 3.1 and 3.2).Gly78 is located in the middle of the long

Figure 3.2. Multiple alignment of the mature part of thermolysin-like proteases. The enzymes listed arefrom B. thermoproteolyticus (TLN, thermolysin; (35)), B. caldolyticus (TLP-cal; (199)), B. stearothermophilusCU21 (TLP-ste; (137)), B. cereus (TLP-cer; (125)), B. megaterium (TLP-meg; (200)), B. brevis (TLP-bre;(201)), B. subtilis (TLP-sub, (30)), B. amyloliquefaciens (TLP-amy; (202)), and elastase from P. aeruginosa((203)). h indicates the glycine residues in the proposed hinge regions. * indicates residues involved in catalysis,Z indicates residues which coordinate the Zn2+. are indicated by light grey boxes. Black boxes indicate residuesinvolved in catalysis. The secondary structure assignment given is that for thermolysin (S, β-strand; T, turn; H,helix; 3, 3 helix).

10

Catalytic hinge-bending motions

33

10 20 30 40 50 60 | | | | | |TLN ITGTSTVGVGRGVLGDQKNINTTYST---YYYLQDN--TRGDGIFTYDA-----KYRTTLPGSLWADADNTLP-cal VAGTSTVGVGRGVLGDQKYINTTYSSYYGYYYLQDN--TRGSGIFTYDG-----RNRTVLPGSLWADGDNTLP-ste VAGASTVGVGRGVLGDQKYINTTYSSYYGYYYLQDN--TRGSGIFTYDG-----RNRTVLPGSLWTDGDNTLP-cer VTGTNKVGTGKGVLGDTKSLNTTLSG--SSYYLQDN--TRGATIFTYDA-----KNRSTLPGTLWADADNTLP-meg VTGTNTIGSGKGVLGDTKSLKTTLSS--STYYLQDN--TRGATIYTYDA-----KNRTSLPGTLWADTDNTLP-bre -----VTATGKGVLGDTKQFETTKQG--STYMLKDT--TRGKGIETYTA-----NNRTSLPGTLMTDSDNTLP-sub ---AAATGSGTTLKGATVPLNISYEG--GKYVLRDLSKPTGTQIITYDL----QNRQSRLPGTLVSSTTKTLP-amy ---AATTGTGTTLKGKTVSLNISSES--GKYVLRDLSKPTGTQIITYDL----QNRENLPGTLVSSTTNQElastase -------AEAGGPGGNQKIGKYTYGSDYGPLIVNDRCEMDDGNVITVDMNSSTDDSKTTPFRFACPTNTY SSSSSSSST T SSSSSSS SSS S T SSSSS3 TTT S S T

70 80 90 100 110 120 130 | h | | | | | |TLN QFFASYDAPAVDAHYYAGVTYDYYKNVHNRLSYDGNNAAIRSSVHYSQGYNNAFWNGSEMVYGDGDGQTFTLP-cal QFFASYDAAAVDAHYYAGVVYDYYKNVHGRLSYDGSNAAIRSTVHYGRGYNNAFWNGSQMVYGDGDGQTFTLP-ste QFTASYDAAAVDAHYYAGVVYDYYKNVHGRLSYDGSNAAIRSTVHYGRGYNNAFWNGSQMVYGDGDGQTFTLP-cer VFNAAYDAAAVDAHYYAGKTYDYYKATFNRNSINDAGAPLKSTVHYGSNYNNAFWNGSQMVYGDGDGVTFTLP-meg TYNATRDAAAVDAHYYAGVTYDYYKNKFNRNSYDNAGRPLKSTVHYSSGYNNAFWNGSQMVYGDGDGTTFTLP-bre YWT---DGAAVDAHAHAQKTYDYFRNVHNRNSYDGNGAVIRSTVHYSTRYNNAFWNGSQMVYGDGDGTTFTLP-sub TFTSSSQRAAVDAHYNLGKVYDYFYSNFKRNSYDNKGSKIVSSVHYGTQYNNAAWTGDQMIYGDGDGSFFTLP-amy FTT-SSQRAAVDAHYNLGKVYDYFYQKFNRNSYDNKGGKIVSSVHYGSRYNNAAWIGDQMIYGDGDGSFFElastase KQVNGAYSPLNDAHFFGGVVFKLYRDWFGT-SPLT--HKLYMKVHYGRSVENAYWDGTAMLFGDGA-TMF S T3 HHHHHHHHHHHHHHHHHHHHHHT T T T SSSSST T S T SSS T

140 150 160 170 180 190 200 hh | Z* Z | * | Z | | | |TLN IPLSGGIDVVAHELTHAVTDYTAGLIYQNESGAINEAISDIFGTLVEFYANKNPDWEIGEDVYTPGISGDTLP-cal LPFSGGIDVVGHELTHAVTDYTAGLVYQNESGAINEAMSDIFGTLVEFYANRNPDWEIGEDIYTPGVAGDTLP-ste LPFSGGIDVVGHELTHAVTDYTAGLVYQNESGAINEAMSDIFGTLVEFYANRNPDWEIGEDIYTPGVAGDTLP-cer TSLSGGIDVIGHELTHAVTENSSNLIYQNESGALNEAISDIFGTLVEFYDNRNPDWEIGEDIYTPGKAGDTLP-meg VPLSGGLDVIGHELTHALTERSSNLIYQYESGALNEAISDIFGTLVEYYDNRNPDWEIGEDIYTPGTSGDTLP-bre LPLSGGLDVVAHELTHAVTERTAGLVYQNESGALNESMSDIFGAMVD-----NDDWLMGEDIYTPGRSGDTLP-sub SPLSGSLDVTAHEMTHGVTQETANLIYENQPGALNESFSDVFGYFND-----TEDWDIGEDITV---SQPTLP-amy SPLSGSMDVTAHEMTHGVTQETANLNYENQPGALNESFSDVFGYFND-----TEDWDIGEDITV---SQPElastase YPLV-SLDVAAHEVSHGFTEQNSGLIYRGQSGGMNEAFSDMAGEAAEFYMRGKNDFLIGYDIKK---GSG T3 3THHHHHHHHHHHHHHHH T T HHHHHHHHHHHHHHHHHHHHHT T ST 3TT T T

210 220 230 240 250 260 | | |* | | |TLN SLRSMSDPAKYGDPDHYSKR----YTGTQDNGGVHINSGIINKAAYLISQGGTHYGVSVVGIGRDKLGKTLP-cal ALRSMSDPAKYGDPDHYSKR----YTGTQDNGGVHTNSGIINKAAYLLSQGGVHYGVSVTGIGRDKMGKTLP-ste ALRSMSDPAKYGDPDHYSKR----YTGTQDNGGVHTNSGIINKAAYLLSQGGVHYGVSVNGIGRDKMGKTLP-cer ALRSMSDPTKYGDPDHYSKR----YTGSSDNGGVHTNSGIINKQAYLLANGGTHYGVTVTGIGKDKLGATLP-meg ALRSMSNPAKYGDPDHYSKR----YTGSSDNGGVHTNSGIINKAAYLLANGGTHYGVTVTGIGGDKLGKTLP-bre ALRSLQDPAAYGDPDHYSKR----YTGSQDNGGVHTNSGINNKAAYLLAEGGTHYGVRVNGIGRTDTAKTLP-sub ALRSLSNPTKYNQPDNYANYRNLPNTDEGDYGGVHTNSGIPNKAAYNTITK----------LGVSKSQQTLP-amy ALRSLSNPTKYGQPDNFKNYKNLPNTDAGDYGGVHTNSGIPNKAAYNTITK----------IGVNKAEQElastase ALRYMDQPSRDGSIDNASQY----YNGI-D---VHHSSGVYNRAFYLLANS--------PGWDTRKAFE S T333TT T3 TT TTTTTH 33HHHHHHHHHHHHH TTTT S HHHHHH

270 280 290 300 310 | | | | |TLN IFYRALTQYLTPTSNFSQLRAAAVQSATDLYGSTSQEVASVKQAFDAVGVK----- 316 TLP-cal IFYRALVYYLTPTSNFSQLRAACVQAAADLYGSTSQEVNSVKQAFNAVGVY----- 319TLP-ste IFYRALVYYLTPTSNFSQLRAACVQAAADLYGSTSQEVNSVKQAFNAVGVY----- 318TLP-cer IYYRANTQYFTQSTTFSQARAGAVQAAADLYGANSAEVAAVKQSFSAVGVN----- 317TLP-meg IYYRANTLYFTQSTTFSQARAGLVQAAADLYGSGSQEVISVGKSFDAVGVQ----- 317TLP-bre IYYHALTHYLTPYSNFSAMRRAAVLSATDLFGANSRQVQAVNAAYDAVGVK----- 304TLP-sub IYYRALTTYLTPSSTFKDAKAALIQSARDLYG--STDAAKVEAAWNAVGL------ 300TLP-amy IYYRALTVYLTPSSTFKDAKAALIQSARDLYG--SQDAASVEAAWNAVGL------ 300Elastase VFVDANRYYWTATSNYNSGACGVIRSAQNRN----YSAADVTRAFSTVGVTCPSAL 301 HHHHHHHHH T THHHHHHHHHHHHHHHHHH THHHHHHHHHHHHH

Chapter 3

34

α-helix (residues 68-87) that crosses the entireN-terminal domain. When studying thestructural effects of zinc substitutions in theactive-site of thermolysin, Holland et al. (150),observed global structural changes thatresembled the previously observed hinge-bending displacement at glycine 78 (86), whichwas taken to confirm the importance of bendingin the 68-88 helix for the overall hinge-bendingmotion.

The studies described above suggest thatconserved glycine residues provide theflexibility required for a catalytic hinge-bendingmotion. In the present study we experimentallyvalidated the importance of these glycineresidues by studying the effects of Gly � Alamutations at various positions in the TLP of B.stearothermophilus CU21 [TLP-ste (152); 86 %overall sequence identity with thermolysin].Five other Gly � Ala mutations were analyzedas controls. Modelling studies were performedto ensure that all alanines could beaccommodated by TLP-ste without theintroduction of atomic clashes or strainedbackbone torsion angles. The effects ofmutations on catalytic activity were evaluatedby determining the specific activities towardscasein and kcat/Km values for threefurylacryloylated synthetic peptides. Mutationaleffects on ligand binding were assessed bydetermining Ki values for the transition-stateinhibitor phosphoramidon. The results provideexperimental evidence for the hypothesis thatconserved glycine residues, in particular Gly78and the Gly135-Gly136 combination, contributeto catalytically important hinge-bendingmotions in TLPs.

Materials and MethodsGenetics.

The nprT gene encoding the TLP of B.stearothermophilus CU21 [TLP-ste, (152)] wascloned, subcloned, and expressed as described

previously (80). Site-directed mutagenesis wasperformed using the pMa/c gapped duplexmethod as described before (80) or (for G78Aand G135A+G136A) by the PCR-based mega-primer method, essentially as described bySarkar and Sommer (139).

Production and characterization of mutantenzymes.

Production, purification, and subsequentcharacterization of the enzymes were performedas described earlier (80), with the exception ofthe G78A mutant, for which a differentpurification protocol was used (82). For thedetermination of thermal stability 0.1 µMsolutions of purified protease (in 20 mM sodiumacetate, pH 5.3, 5 mM CaCl2, 0.01% Triton X-100, 0.5% 2-propanol, and 62.5 mM NaCl) wereincubated at various temperatures for 30 min,after which the residual proteolytic activity wasdetermined using casein as a substrate (136).Thermal stability was quantified by T50, beingthe temperature giving 50% residual proteaseactivity after a 30 min period of incubation.Wild-type TLP-ste (T50 = 73.4 °C) was includedin every assay and thermal stabilities of mutantsare presented by the change in T50 compared towild-type TLP-ste (δT50).

The specific activity of the TLPs oncasein was determined according to a methodadapted from Fujii et al. (136): approximately0.5 µg of protease was incubated in 1 ml of 50mM Tris-HCl, pH 7.5, containing 0.8 %(wt/vol) casein and 5 mM CaCl2 at 37 °C for 1h. The reaction was quenched by adding 1 ml ofa solution containing 100 mM TCA, pH 3.5.The specific activity was calculated as theaverage from at least three independent assays.One unit of activity is defined as the amount ofenzyme activity needed to liberate a quantity ofacid-soluble peptide corresponding to anincrease in A275nm of 0.001 per min.

Catalytic hinge-bending motions

35

The catalytic performance (kcat/Km) forthree furylacryloylated peptides at 37 °C wasdetermined essentially as described by Feder(114) using a thermostatted Perkin-ElmerLambda 11 spectrophotometer. The reactionmixture (1 ml) contained 10 mM MOPS-NaOH,pH 7.0, 5 mM CaCl2, 5 % Me2SO (v/v), 1 % 2-propanol (v/v), 0.02% Triton-X100 (v/v), 125mM NaCl, 100 µM substrate, and varyingamounts of enzyme. Activities were derived bymeasuring the decrease in absorption at 345 nmusing a ∆ε of -317 M-1cm-1 as described byFeder (114).

The furylacryloylated peptides 3-(2-furylacryloyl)-L-glycyl-L-leucine amide(FaGLa), 3-(2-furylacryloyl)-L-alanyl-L-phenylalanine amide (FaAFa) and 3-(2-furylacryloyl)-L-glycyl-L-leucinyl-L-alanine(FaGLA) were obtained from BachemFeinchemikalien AG, Bubendorf, Switzerland.

The Ki for phosphoramidon (N-[α-L-rhamnopyranosyl-(oxyhydroxyphosphinyl)]-L-leucyl-L-tryptophan; Boehringer Mannheim,Germany) was determined by a 30 minpreincubation of 100 pM protease with varyingconcentrations of the inhibitor (10-8 to 10-5), in abuffer consisting of 50 mM Tris-HCl, pH 7.5, 5mM CaCl2, 5% Me2SO (v/v), 1% 2-propanol(v/v), and 125 mM NaCl, after which FaAFa(final concentration 100 µM) was added assubstrate. Ki s were calculated by the methoddescribed by Hunter & Downs (145).

Structural analysis.A three-dimensional model of TLP-ste

was built on the basis of the crystal structure ofthermolysin, using the molecular modellingprogram WHAT IF (140). The modellingprocedures have been described in detailelsewhere (78). Because of the 86 % sequenceidentity between the template and the model, theTLP-ste model was expected to be sufficientlyreliable to predict and analyze the effects of

site-directed mutations (78, 141). This idea wascorroborated by the successful de novo designof many stabilizing mutations (e.g., (43, 78,82)). In this report all TLPs are numberedfollowing the corresponding residues inthermolysin. All glycine residues mutated in thisstudy have Φ, Ψ angles that are favorable foralanine (see http://swift.EMBLHeidelberg.DE/neutpep/ for structural details). The Φ, Ψangles of Gly89 and Gly109 are in the left-handed α-helix region. The feasibility ofmutating these was, however, apparent fromprevious studies (153) and from the fact thatresidues other than glycine do occur at thesepositions in TLPs from other bacilli.

ResultsSelection of mutations and production of mutantproteins.

In addition to TLP-ste variants withGly�Ala mutations at positions 78, 135, and136 (and the 135/136 double mutant), five otherTLP-ste variants, each containing one Gly�Alamutation were included in this study. Four ofthese 'control' mutations were in parts of theprotein (positions 58, 89, 109, and 264) that areunrelated to catalysis or hinge-bending (Fig.3.1). The fifth (at position 141) is located nextto His142, which is part of the HExxH motifcommon to all zinc metalloproteases [Fig 3.1and 3.2; see also (154)]. The two histidines inthis motif are ligands of the active-site zinc andthe glutamate is directly involved in thecatalytic mechanism (92, 155). The C-β ofAla141 points away from the active-site and ishighly unlikely to influence the binding of theligand.

Modelling studies indicated that themutations at positions 78, 135, and 136 wouldnot affect substrate binding by direct contactsbetween the introduced alanine C-β and the

Chapter 3

36

Catalytic hinge-bending motions

bouTheclasIle1Phe3.3resuPhebinpheHothetheof phoproexcG15−8theweron

Figure 3.3. Local environment of in the hinge area of the G135A+G136A mutant of TLP-ste. There isclearly some van der Waals overlap between the C-β of Gly136Ala and the C-δ of Ile192, which again could

37

nd ligand (nor would the other mutations). model indicated a minor Van der Waalsh between the C-β of Ala136 and the C-δ of92, which interacts with the side chain of133 at the bottom of the S1' pocket (Fig.

). The G136A mutation could, therefore,lt in a small displacement of the Ile192 and133 side chains, which, in turn could disturbding of substrates with large side chains (e.g.nylalanine) in the P1' position (Fig. 3.3).wever, modelling studies and inspection of crystal structure of the phosphoramidon-rmolysin complex (52) indicated that the C-β

Ala136 would not affect binding ofsphoramidon. All mutant proteins could beduced in standard amounts, with theeption of the G78A mutant and the35A+G136A double mutant, that yielded-fold less protease upon fermentation. With

exception of the G78A mutant, all mutantse purified using the standard protocol basedaffinity chromatography with bacitracin-

silica (80, 144). The G78A mutant did not bindefficiently to the bacitracin-silica column and adifferent purification method (82), was thereforeemployed to purify this mutant.

Effects on activity.The mutant proteins were characterized

by determining specific activities towardscasein, kcat/Km values for variousfurylacryloylated peptides (114), and Ki valuesfor the transition state inhibitor phosphoramidon(52) (Table III.I). Since the low solubilities ofthe furylacryloylated peptides preclude accuratedetermination of Km values (114, 115, 133), theKi's phosphoramidon were determined toexamine whether the mutations had affectedligand binding.

For the control mutations (G58A, G89A,G109A, G141A, and G264A), both increasesand decreases in catalytic activity of, at most, afactor of 2 were observed. These small effectswere substrate-dependent, being only detectable

affect Ph133 at the bottom of the S1' subsite.

Chapter 3

38

for one or two of the substrates tested (TableIII.I). Clearly, larger effects were observed forG78A, G136A, and G135A+G136A. Theseeffects were negative in all cases and they wereobserved for all peptide substrates tested.Depending on the peptide used, G78A, G136A,and G135A+G136A reduced kcat/Km 4−6-fold,2−5-fold, and 8−40-fold, respectively.Remarkably, clear effects on specific activitytowards casein were only observed for G78A(Table III.I). The G135A+G136A doublemutation reduced activity towards casein only2-fold, which is a reduction in the same order ofmagnitude as for one of the control mutations.

The G135A single mutant behaved likewild-type enzyme for all substrates tested.Nevertheless, G135A+G136A was much lessactive than the mutant containing G136A alone.This suggests that the flexibility provided byG135A is not essential for activity as long asthere is a glycine present at position 136.

As predicted from the modelling studiesdescribed above, G136A affected substratespecificity to some extent. This is shown by asmall reduction in the [kcat/Km (FaAFa)]/[kcat/Km

(FaGLa)] ratio observed for G136A andG135A+G136A (Table III.I). Wild-type TLP-ste and G136A containing mutants gave almostidentical digestion patterns when incubated withβ-casein as a substrate, indicating that theeffects of G136A on cleavage specificity indeedwere small [B. van den Burg and O.R. Veltman,unpublished observations; method described in(84)]. Interestingly, the effects of G136A andG135A+G136A were dependent on the lengthof the substrate used. This is illustrated by theincrease in the [kcat/Km (FaGLA-OH)]/[kcat/Km

(FaGLa)] ratio in the double mutant. For evenlonger substrates such as casein, hardly anydecrease in activity of the G136A and theG135A+G136A mutant enzymes wasdetectable. The effects of G136A andG135A+G136A on the Ki for phosphoramidon

were small in comparison with the effects onkcat/Km (Table III.I). Since the present studydoes not involve mutation of residues directlyinteracting with ligands/susbstrates, it isreasonable to assume that effects on the Ki tosome extent reflect effects on binding affinity ingeneral. The modest effects on Ki thus indicatethat the larger effects on kcat/Km are primarilydue to effects on kcat and not to effects on Km.The fact that the G136A and G135A+G136Amutants behaved as the wild-type enzymeduring affinity chromatography supports thisconclusion.

The effects of G78A were approximatelysimilar for all peptide substrates as reflected bythe grossly unchanged ratios given in TableIII.I. Thus this mutation did not affect or hardlyaffected substrate specificity, nor were theobserved reductions in activity substantiallydependent on substrate-length. Consistent withthe latter observation, the effects of G78A arealso reflected in reduced activity towards casein.The increase in Ki for phosphoramidon (whichis in the same order of magnitude as thedecrease in the various kcat/Km values; TableIII.I) and the strong reduction in binding tobacitracin indicate that a significant part of thereduction in catalytic efficiency caused byG78A reflects an increase in Km and thus areduced ability to bind substrate.

Effects on stability.Based on the results of statistical (156,

157), theoretical (158) and experimentalanalyses [e.g. (159-163)] Gly�Ala mutationsare expected to be beneficial for proteinstability, albeit in a context dependent way (162,163). In contrast with this notion, most of theGly�Ala mutations presented in this study hadonly marginal effects on stability, with theexception of G58A (stabilization by 3.8°C) andG78A (destabilization by 5.5°C) (Table III.II).Interestingly, the rather drastic (in terms of

Catalytic hinge-bending motions

39

activity) G135A+G136A double mutationhardly affected protein stability.

DiscussionStudies of the crystal structures of

enzymes in the ligand-free and ligand-bindingstate provide accumulating evidence for thenotion that larger, concerted motions are anessential part of catalysis in many enzymes(164). A hinge-bending movement betweendomains that close around an enzyme's active-site is one, relatively simple example of suchconcerted motions (86, 88). Studies of a largenumber of wild-type and mutant T4 lysozymestructures (87, 88, 165) strongly indicated thathinge-bending motions resulting in continuousopening and closure of the active-site cleft arean intrinsic property of this enzyme.Interestingly, Mchaourab et al. (166) haverecently been able to demonstrate the hinge-bending motion in T4 lysozyme in solution bydirectly measuring inter-residue distances withhelp of site-directed spin-labeling. Hinge-bending motions have also been detected bymolecular dynamics simulations of variousproteins (151, 167) including thermolysin (89).These motions were similar to the motionsinferred from structure comparisons (86). Since

domain closure is supposedly related toentrapment of substrate and, thus, to catalysis,the hinge-bending motion needs to be fast (164).Energy barriers for torsion angle variations thusneed to be low, which would be achieved best inthe case where the hinge residue is a glycine.The latter is strongly supported by the presentresults, which show that Gly�Ala mutations inthe hinge regions, but not those at five controlpositions, drastically reduce enzymatic activity.

Inspection of the TLP alignment (Fig.3.2) shows that, in addition to positions 135 and136, there are several other positions in andaround the central interdomain helix (137-150)where glycines are conserved. The absence of aglycine at position 136 is correlated with thepresence of a glycine at position 147, at theother end of the central α-helix. This Gly147has been changed into an alanine by Margarit etal. (161) in an attempt to stabilize the TLP fromB. subtilis (Gly135, Ser136, Gly147).Interestingly, like the Gly136A mutant in TLP-ste, the Gly147Ala mutant displayed reducedactivity towards peptide substrates, whereas theactivity towards casein was hardly affected.Elastase lacks both Gly135 and Gly136, but thismay be compensated by glycines at positions147 and 154. Both of these glycines areconserved in almost all more distantly relatednon-Bacillus TLPs which lack Gly135 andGly136 (not shown; see http://swift.EMBL-Heidelberg.DE/neutpep/ ). It should be notedthat, in addition to permitting low energy-barrierconcerted motions, the various glycines in TLPactive-sites (135, 136, 141, 147, 154; see Fig.3.2) are likely to contribute to the localflexibility in the active-site, that is generallyconsidered to be important for catalysis (106,168). It has recently been claimed that theactive-sites of many enzymes containrecognizable, glycine containing sequencemotifs (169).

Some of the control mutations (58, 141,264) in this study had small but significant

Table III.II. Thermal stability of glycine→→→→alanineTLP-ste variants.

variant δT50a,b variant δT50a, b

°C °CTLP-ste 0 G135A -0.3G58A +3.8 G136A +0.1G78A -5.5 G135A+G136A -0.6G89A 0 G141A +0.7G109A 0 G264A +0.2

a wild-type T50=73.4 °C. Part of the effects on thermalstability have been discussed previously (78, 154).bδT50 values are relative to wild-type; the standarddeviations in the δT50 measurements were below± 0.3°C in all cases.

Chapter 3

40

effects on catalytic activity towards the peptidesubstrates. The negative effect of G141A is notsurprising since it may directly affect theconformation and flexibility of crucial parts ofthe active-site (154, 169). The negative effect ofG58A and the positive effect of G264A are lessreadily explainable. The effects of the controlmutations were in all cases much smaller thanthe effects of G78A or the G135A, G136Adouble mutation.

As shown in Table III.II, most Gly�Alamutations had only marginal effects on thermalstability. Previously, it has been shown thatthermal inactivation of TLP-ste is determinedby (rate-limiting) partial unfolding processes,followed by autolytic degradation (80, 170,171). Consequently, only mutations in regionsthat are involved in the these partial unfoldingprocesses are expected to affect stability. InTLP-ste such a region was identified in the N-terminal domain, in particular between residues4 and 70 (77, 78, 82, 153). Gly58 is animportant part of this region and the stabilizingeffect of the G58A mutation has been discussedpreviously (153, 172). Modelling studies haveindicated that the destabilizing effect of G78Amight be caused by a clash between the alanineC-β and the N-η1 of Arg35, which woulddisturb a cluster of electrostatic interactionsinvolving several residues in the stabilitydetermining region (Arg35, Asp32, Asp85; notshown).

So far, the various types of studies of the(presumed) catalytic hinge-bending motion inTLPs (54, 86, 89, 127, 150) provide a highlyconsistent picture, showing that, indeed, such amotion occurs. However, although residues 78,135 and 136 appear to be important for the samecatalytic hinge-bending motion, the effects ofmutations at these positions differ. G78Areduced activity towards all substrates tested,whereas G135A+G136A reduced activitytowards shorter substrates only. Furthermore,

the data may be taken to indicate that the kcat

component in the mutational effect is relativelysmall for G78A and relatively large forG135A+G136A. At the moment we can onlyspeculate about the cause of these differences. Itis conceivable that mutations at position 78 havea more general effect on mobility in thesubstrate binding cleft, whereas mutations at135 and 136 (which are at the beginning of the'catalytic' inter-domain helix) primarily affectmotions directly involved in catalysis. In theirdetailed structural studies of hinge-bendingdisplacements in thermolysin, Holland et al.(150) concluded that closure of the active sitemay be directly related to optimizing theenzyme for binding of the transition state, thuslinking the hinge-bending motion to kcat. Forlonger substrates, the contribution of bindingenergy to catalysis (173) may be so large thatthe negative effect of the G135A+G136Adouble mutation on the catalytic rate becomesrelatively small and hardly detectable. Thedistortion (kink) in the 68-88 helix that ispresumably needed to "open" the active-site ofthermolysin [see model presented in (150)] maybe less easy to achieve when glycine at 78 hasbeen replaced by alanine. Thus, G78A may have"locked" the enzyme in a more closed stateresulting in reduced ligand affinity. In thisrespect, it is suggestive that the G78A mutant isthe only TLP-ste mutant (out of severalhundreds made) that did not bind to bacitracin-silica during purification. Insight in the cause ofthe differences in the mutational effects as wellas increased understanding of the hinge-bendingmotion may be gained by further enzymologicaland crystallographic analyses of the mutantsdescribed above.

AcknowledgementsThe authors like to thank Daan van

Aalten for helpful discussions and Ype van derZijpp for help with some of the experiments.

S1' subsite mutations

41

The effect of changing the hydrophobic S1'subsite of thermolysin-like proteases on substrate

specificity.

AbstractThe hydrophobic S1' subsite is one of the

major determinants of the substrate specificityof thermolysin and related M4 family proteases.In the thermolysin-like protease (TLP) producedby B. stearothermophilus (TLP-ste), thehydrophobic S1' subsite is mainly formed byPhe130, Phe133, Val139 and Leu202. In thepresent study, we have examined the effects ofreplacing Leu202 by smaller (Gly, Ala, Val) andlarger (Phe, Tyr) hydrophobic residues. Themutational effects showed that the wild-type S1'pocket is optimal for binding leucine sidechains. Reduction of the size of residue 202resulted in a higher efficiency towardssubstrates with Phe in the P1' position. Ratherunexpectedly, the Leu202Phe and Leu202Tyrmutations, which were expected to decrease thesize of the S1' subsite, resulted in a largeincrease in activity towards dipeptide substrateswith Phe in the P1' position. This is probablydue to the fact that 202Phe and 202Tyr adopt asecond possible rotamer which opens up thesubsite compared to Leu202 and which favoursinteractions with the substrate. To validate theseresults we constructed variants of thermolysinwith changes in the S1' subsite. Thermolysinand TLP-ste variants with identical S1' subsiteswere higly similar in terms of their preferencefor Phe versus Leu in the P1' position.

IntroductionThermolysin-like proteases (TLPs) are

members of the peptidase family M4 (51) ofwhich thermolysin (TLN, EC .3.4.24.27) is theprototype. The amino acid sequences of severalTLPs have been determined [see (51), or the

Merops data base at http://www.merops.co.uk/merops/famcards/m4.htm], and the three-dimensional structure of TLPs isolated fromseveral bacteria have been solved [Bacillusthermoproteoliticus (52), Bacillus cereus (54,54), Pseudomonas aeruginosa (55) andStaphylococcus aureus (56)]. TLPs consist of anα-helical C-terminal domain and an N-terminaldomain mainly consisting of β-strands. Thedomains are connected by a central α-helix.This helix is located at the bottom of the activesite cleft and contains several of the catalyticallyimportant residues (Fig. 4.1). Four substratebinding pockets [S2, S1, S1' and S2';nomenclature according to Schechter andBerger (174)] have been identified (62). The S1'subsite is a hydrophobic pocket which isconsidered a major determinant of substratespecificity (114, 133). In thermolysin and theTLP produced by B. stearothermophilus (TLP-ste), the subjects of this study, the S1' subsite ismainly formed by Phe130, Val139, Leu202 andPhe133 (TLN) or Leu133 (TLP-ste).

Crystallographic (52, 61, 62, 155) andmodelling studies (62) of thermolysin haveindicated that the S1' subsite allows efficientbinding of a leucine side chain. The notion thatthe S1' subsite in thermolysin is not optimal forbinding larger residues such as phenylalanine(61, 62) was experimentally confirmed byIzquierdo and Stein (175). These authorsshowed a clear positive correlation between thesize of the P1' residue and the activity of theenzyme on dipeptide substrates of the 3-(2-furylacryloyl)-L-glycyl-L-Xxx-amide type(FaGXa, where Xxx is a hydrophobic aminoacid). Phenylalanine, however, did not conform

Chapter 4

42

to this trend as illustrated by the fact that similarkcat/Km values were obtained for FaGLa andFaGFa. The S1' subsite of TLP-ste is similar instructure and character to that in thermolysin,but TLP-ste has a higher preference forsubstrates with a Phe at P1' .

In the present study we haveinvestigated the possibility of modifying the S1'subsite in TLPs in order to change thepreferences of the enzyme for Leu and Phe inthe P1' position. Our hypothesis was that alimited increase in the size of the S1' pocketcould result in an enzyme that would retain itscatalytic power, while displaying an increasedpreference for Phe at position P1' in thesubstrate.

As we have shown previously (176),mutating Phe133Leu results in a decreasedspecificity for P1' Phe substrates in TLP-ste andchanges its substrate specificity into that ofTLN. We therefore chose to mutate residueLeu202 which, together with residue 133dominates the entrance of the substrate to theS1' pocket (62, 176). We were particularlyinterested in TLP-ste since we have previouslyconstructed a highly thermostable variant of thisenzyme (84). To validate our models of TLP-steand the S1' pocket of the M4 peptidases ingeneral, we also constructed and characterized anumber of mutants in thermolysin.

Our results show that it is indeedpossible to increase the preference for a P1' Pheby mutating residue 202. The results also

Leu202

Figure 4.1. Ribbon diagram of thermolysin with a substrate in the active site clef. The left side shows theN-terminal domain consisting predominantly of β-sheet; the right side the predominantly α-helical C-terminaldomain. The central α-helix, at the bottom of the active site cleft, contains several residues important forcatalysis, including those coordinating the Zn2+ ion (blue sphere). The substrate is the tripeptide Gly-Phe-Ala,occupying the S1 to S2' positions. The Gly is modeled, whereas the Phe-Ala part is taken from one of thethermolysin structures. The S1' side chains shown are (counter clockwise, starting at 202Leu); 202Leu, 130Phe,133Leu and 139Val.

S1' subsite mutations

43

indicate that the substrate specificity of TLP-steand TLN will change in a similar manner tomutations at positions 133 and 202. Thissupports the idea that the specificity of other M4peptidases can be changed in the same way as inTLP-ste and TLN.

Materials and MethodsModelling and mutant design.

A three-dimensional model of TLP-stewas built on the basis of the crystal structure ofthermolysin, using the molecular modellingprogram WHAT-IF (140), as has been describedelsewhere (78). The high sequence identitybetween thermolysin and TLP-ste (86%)indicates that the TLP-ste model should besufficiently reliable for prediction and analysisof the effects of most amino acid substitutions(78, 141). Indeed, the TLP-ste model has beenused successfully for the design of variousstabilizing mutations (43, 82, 142, 177).Throughout this paper, residues in all TLPs arenumbered according to the numbering ofcorresponding residues in thermolysin.

Molecular biological techniques.The nprT gene encoding the TLP of

Bacillus stearothermophilus CU21 (136) (TLP-ste) was cloned, subcloned, and expressed asdescribed previously (80). The plasmidpUBTZ2 (171) containing the nprM geneencoding thermolysin (135) was obtained fromDSM-HSC, Geleen, The Netherlands. Site-directed mutagenesis was performed either bythe PCR-based mega-primer method, essentiallyas described by Sarkar and Sommer (139) orwith the QuikChange site-directed mutagenesiskit from Stratagene, La Jolla, USA. TheQuickChange procedure basically uses a pair ofcomplementary PCR primers that places themutation in the middle of the primers. pUC18containing a subcloned fragment of nprT, orpUBTZ2 containing nprM, was amplified usingPyrococcus furiosus DNA polymerase (Pfu

Turbo) and these primers for 18 cycles in aDNA thermal cycler. After digestion of theparental DNA with DpnI, the amplified DNAincorporated with the nucleotide substitutionwas transformed into E. coli XL1-Blue strain.Mutagenic primers were designed such thatmutant clones could be recognized by thepresence or absence of an endonucleaserestriction site (80). The nucleotide sequencescoding for the mature part of the proteases wereverified by DNA sequence analysis. Themutated fragments of TLP-ste weresubsequently cloned into the Bacillus expressionvector pGE501 (178) containing the TLP-stegene with a deletion of the previously subclonedfragment.

Production and Characterization of MutantEnzymes.

Production and purification of theenzymes were performed as described earlier(80) using the B. subtilis strain DB117(∆npr,∆apr)(144). Before determining thekinetic parameters, protease preparations weredesalted using pre-packed PD-10 gel filtrationcolumns supplied by Amersham Pharmacia,Uppsala, Sweden. Specific activities of theTLP-ste variants towards casein weredetermined according to a method adapted fromFujii et al. (136): approximately 0.5 µg ofprotease was incubated in 1 ml of 50 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol(Tris⋅HCl), pH 7.5, containing 0.8% (wt/vol)casein and 5 mM CaCl2 at 37°C for 1 h.Reactions were quenched by the addition of 1ml of a solution containing 100 mM tri-chloro-acetic acid (TCA), pH 3.5. One unit of activitywas defined as the amount of enzyme activityneeded to liberate a quantity of acid-solublepeptide corresponding to an increase in A275nm

of 0.001 per minute.The kcat/Km and Km values of the

enzymes for furylacryloylated di- and

Chapter 4

44

tripeptides were determined at 37°C, in athermostated Perkin-Elmer Lambda 11spectrophotometer. The reaction mixture (1 ml)contained 50 mM Tris, 50mM 4-morpholineethanesulfonic acid (MES), pH 7.0,5 mM CaCl2, 5% Me2SO, 0.5% 2-propanol,0.01% Triton X-100 and 100 µM to 2.5 mM ofsubstrate, and the reaction was followed bymeasuring the decrease in absorption at 345 nm(∆ε345 = -317 M-1⋅cm-1 )(179). All substrateswere supplied by Bachem AG, Bubendorf,Switzerland. Stock solutions of thefurylacryloylated dipeptides 3-(2-furylacryloyl)-L-glycyl-L-leucine-amide (FaGLa) and 3-(2-furylacryloyl)-L-glycyl-L-phenyl-amide(FaGFa), and of the furylacryloylatedtripeptides 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine (FaGLA) and 3-(2-furylacryloyl)-L-glycyl-L-phenylalanine-L-leucine (FaGFL) were prepared by dissolvingthe peptides in Me2SO. Apparent second orderrate constants (kcat/Km) were determined byvarying the enzyme concentrations over a 50-fold range under pseudo-first-order conditionsand measuring the initial activity, essentiallyaccording to the method described by Feder(179).

The Ki for phosphoramidon (N-[α-L-rhamnopyranosyl-oxyhydroxyphosphinyl]-L-leucine-L-tryptophan), Roche MolecularBiochemicals, was determined by a 10 minutespreincubation of a 0.1 nM protease solutionwith varying concentrations of the inhibitor (10-

8 to 10-3 M), in 50 mM Tris, 50 mM MES, pH7.0, 5 mM CaCl2 , 0.01% Triton X100.Subsequently, enzyme activity was determinedusing 100µM FaGLA as substrate. Ki ’s werecalculated by the method described by Hunterand Downs (145).

ResultsMutant design and production of mutantproteins.

Position-specific rotamer searches (180)for the residues to be introduced at position 202and 133 showed that all new side chains couldadopt a favorable rotamer without concomitantintroduction of steric overlap in both TLP-steand thermolysin. Furthermore, the modellingstudies indicated that the Leu202Gly,Leu202Ala and Leu202Val mutations wouldsimply increase the size of the pocket by avolume corresponding to approximately four,three and one methyl groups, respectively,whereas Leu202Phe and Leu202Tyr wereexpected to lead to some reduction in size.

The various mutant TLPs wereconstructed, produced, purified andcharacterized as described in Materials andMethods. Production and purification yieldswere similar to those of the wild-type TLP-ste.All variants had similar thermal stabilities andspecific activities towards casein (data notshown).

Characterization of mutant proteases.To examine the enzymatic properties of

the mutant TLP-ste enzymes, kinetic parametersfor the reaction with available tripeptidesubstrates as well as the Ki for phosphoramidonwere determined (Table IV.I). The kcat/Km

values for FaGLA indicate that leucine is theoptimal residue at position 202 for substrateswith a Leu at the P1' position. The results withFaGFL show that the activity towards substrateswith a phenylalanine at the P1' position wasincreased by replacing Leu202 by a smallerresidue. For FaGFL, the Leu202Ala mutant wasthe most active. It is interesting to note that theLeu202Phe and Leu202Tyr mutations weremore deleterious for activity towards FaGLAthan for activity towards FaGFL (with the largerPhe at P1' ). The Tyr variant was more activethan the Phe variant for both substrates.

S1' subsite mutations

45

Chapter 4

46

The solubility of the tripeptide substrates wassufficient to allow determination of the kcat andKm values for TLP-ste variants. A complicationof the use of tripeptides is that they also occupythe S2' pocket. The S2' pocket could beaffected by mutation of Leu202 (62), meaningthat the mutational effects displayed in TableIV.I may in part be due to effects on binding ofthe P2' residue in the substrate. The Morihara-Tsuzuki specificity order (115) shows thatsubstrates with a P2' Leu (as in FaGFL) have ahigher affinity and a higher kcat/Km thansubstrates with a P2' Ala (as in FaGLA). Sincethe available tripeptide substrates do not havethe same P2' residue, interpretation ofmutational effects in terms of changedpreferences for the P1' residue is notstraightforward. Therefore, additionalcharacterization of the TLP-ste variants wasconducted using the dipeptide substrates FaGLaand FaGFa. Because of low solubilities of thesesubstrates, only kcat/Km values could bedetermined (Table IV.II). In accordance withthe results obtained with tripeptide substrates,replacement of Leu202 with a smaller residueresulted in a decrease in kcat/Km for FaGLa andan increase in kcat/Km for FaGFa, thus yielding adrastic overall change in the preferences forleucine and phenylalanine at P1'. Replacement

of Leu202 by Phe or Tyr had only modesteffects on activity towards FaGLa, inaccordance with observations with the tripeptideFaGLA. Most remarkably, replacement ofLeu202 by Phe or Tyr resulted in a spectacularincrease in the kcat/Km for FaGFa.

The high sequence identity betweenTLP-ste and thermolysin (86 % ) ensures thatthe three-dimensional model of TLP-ste is quiteaccurate, especially in well-conserved regionssuch as the active site [e.g. see (78, 141, 181)for a discussion]. To verify the presumedsimilarity of thermolysin and TLP-ste withrespect to the S1' subsite, thermolysin wasmutated such as to make its S1' subsite"identical" to that of some of the TLP-stevariants. The subsite in both enzymes iscomposed of Phe130, Val139, Leu202 andPhe133 in TLP-ste or Leu133 in thermolysin.As shown in Table IV.III, TLN and TLP-stevariants with identical residues at positions 130,133, 139, and 202 had virtually similarpreferences for substrates with Phe versus Leuat the P1' position. This adds confidence to thenotion that the S1' subsites of TLN and TLP-steare structurally similar and that the model ofTLP-ste can be used to design mutations in thissubsite.

Table IV.II. Specificity of B. stearothermophilus thermolysin-like protease (TLP-ste) variants fordipeptide substrates.

FaGLa FaGFa FaGFakcat/Km

a,b kcat/Km kcat/Kma,b kcat/Km FaGLa

s-1⋅M-1 ×10-3 relative s-1⋅M-1 ×10-3 relative ratio kcat/Km

L202G 0.2 10% 3.3 97% 14.71L202A 0.5 22% 27.8 810% 56.39L202V 2.2 97% 12.7 370% 5.88TLP-ste 2.2 100% 3.4 100% 1.55L202F 0.9 42% 11.8 344% 12.62L202Y 3.1 141% 56.6 1648% 18.12

aReaction conditions: 50 mMTris, 50 mM MES, 5 mM CaCl2, 0.01% Triton X100, pH 7.0, 37°C.bStandard deviations are less than 15% of the value given.

S1' subsite mutations

47

DiscussionA comparison of the results obtained for

thermolysin and TLP-ste shows that theseenzymes have virtually identical substratepreferences (expressed in the ratio's of kcat/Km)if they contain identical residues at positions133 and 202. This shows the reliability of theTLP-ste model (at least near the S1' subsite) andthe usefullness of thermolysin crystal structuresin discussing the effects of the mutations. Theactivities of the different enzymes towards eachsubstrate show that thermolysin generally is amore active enzyme than TLP-ste. Although wecan only speculate as to the origin of thisdifference, differences in hinge-bendingmotions (86, 148) or in active site electrostaticsattributable to the 42 dissimilarities in aminoacid composition may play a role.

Owing to extensive crystallographicstudies by Matthews and co-workers (52, 61,155), considerable information is availableconcerning the interaction between thermolysinand a variety of ligands. Superposition of aseries of enzyme-ligand complexes shows thatthe residues making up the S1' subsite havehighly invariant positions (0.15 – 0.20 Å rms.),

indicating that they hardly adapt to the P1'residue in the ligand (Fig. 4.2). Instead, theligand seems to adapt, thus ensuring thatspecific ligand-enzyme interactions arepreserved, regardless of the type of P1' residue.Ligands with a leucine chain at P1' show aprominent preserved interaction involving a Cδatom on the substrate and a Leu133-Cδ and aLeu202-Cδ in the S1' pocket. In case ofshorter P1' side chains without Cδ, the Cαatoms of the ligand shift (by up to 1.2 Å).Consequently, a carbon in the P1' side chainoccupies the position which is occupied by oneof the Cδ atoms in case P1' is leucine. Thus,contrary to what might be expected, largersubstrates do not penetrate the pocket deeperthan smaller substrates. In addition to showingthat the Cδ atoms of Leu202 have importantinteractions with the P1' side chain, the crystalstructures also suggest that these Cδ atoms are amajor sterical hindrance for accommodatinglarger side chains (such as Phe) in the S1'pocket.

Almost any mutation of Leu202 reducedthe activity towards substrates with Leu at P1',

Table IV.III. Comparison of the specificity of thermolysin and TLP-ste mutants towardsdipeptide substrates.

Position kcat/Kma,b Phe

133 202 FaGLa FaGFa Leus-1⋅M-1 ×10-3 s-1⋅M-1 ×10-3 ratio kcat/Km

TLN wt Leu Leu 12.3 3.9 0.3Phe Leu 10.8 15.6 1.5Leu Tyr 25.3 230.0 9.1Phe Tyr 13.0 186.4 14.3

Leu Leu 17.2 6.3 0.4TLP-ste wt Phe Leu 2.3 3.4 1.5

Leu Tyr -- -- --Phe Tyr 3.1 56.6 18.3

aReaction conditions: 50 mMTris, 50 mM MES, 5 mM CaCl2, 0.01% Triton X100, pH 7.0, 37°C.bStandard deviations are less than 15% of the value given.

Chapter 4

48

whicthe optimThusresula P1'

incresubstof remutatowastudibest creatLeu2muchchain

rotam

Figure 4.2. The P1' residues preferentially occupy a specific location in the S1' subsite. A line diagramshowing a superposition of several thermolysin structures (1TLP.PDB, 3TMN.PDB, 2TMN.PDB, 6TMN.PDB,1TMN.PDB and 4TLN.PDB). The arrow indicates the position that is always occupied by a ligand-carbon atom(see text for details). The superposition shows the remarkable rigidity of the subsite residues and also shows thevariability in the positions occupied by the various ligands.

h is in accordance with the notion that inwild-type enzyme the 202 residue isized for binding a Leu in the S1' pocket.

, any other residue at the 202 position willt in lower kcat/Km values for substrates with Leu (62, 175).

Interestingly, it was indeed possible toase the activity of TLP-ste towardsrates with Phe at P1' by reducing the sizesidue 202. Of the Val, Ala and Glynts, the Ala mutant was most activerds substrates with a Phe at P1'. Modellinges (not shown) indicated that alanine is thecompromise between on the one hand

ing space in the S1' pocket (not sufficient in02Val) and on the other hand keeping as as possible contacts with the P1' side (better in Leu202Ala than in Leu202Gly).

If Phe and Tyr would adopt the sameer as Leu at position 202, the size and

accessibility of the S1' pocket would bedrastically decreased. This would obviouslylead to a drastic reduction in catalytic activitytowards all substrates tested. Such drasticreductions were not observed, indicating thatPhe and Tyr adopt a second favorable side chainconformation in which the aromatic ring isparallel to the substrate. Molecular modellingindicates that the distance between the aromaticrings of 202Phe/Tyr and the P1' Phe wouldthen be approximately 4 Å, which is appropriatefor hydrophobic packing but too large foraromatic stacking.

The Tyr mutant had clearly anomalouscharacteristics, such as a drastically increasedKi, and higher activity than the Phe mutant forall substrates tested. Most remarkably, the Tyrmutant clearly had the highest activity towardsFaGFa of all variants tested in this study.Modelling studies indicate that this anomalous

S1' subsite mutations

49

behavior might be caused by a hydrogen bondbetween the Tyr-OH and the substrate. Adetailed explanation of the remarkable effects ofLeu202Tyr awaits crystallographic studies ofthe mutant.

In case of substrates with a Phe at P1',mutational effects were more pronounced forthe dipeptides than for the tripeptides. This maybe due to the fact that residue 202 also affectsthe S2' subsite [illustrated by thermolysin-ligandcomplexes with PDB accession numbers 1TLP,5TMN and 6TMN (182-184)]. It would not besurprising if effects on the S2' subsite are mostnotable for FaGFL, since this substrate has aLeu at P2'. As explained above, a Leu at P2'interacts strongly with S2' (115) and itsinteractions with the enzyme are thus mostlikely to be affected by mutation of residue 202.

Several mutagenesis studies of proteaseshave shown that it is possible to manipulatesubstrate preferences by changing the sizeand/or character of hydrophobic binding pockets(95, 96, 98, 113, 185-190). For example,diminishing the space in the S1 subsite ofsubtilisin YaB (95) and subtilisin E (185) byincreasing the size of the subsite residues led toreduced activity towards substrates with large P1

residues while yielding higher activity towardssubstrates with smaller P1 residues. When it

comes to binding of hydrophobic side chains ina subsite, substrate and subsite geometry play arole in addition to subsite size [e.g. (96, 113)].In the present study we probably see both typesof effects. The activity effects of reducing thesize of residue 202 are likely to be caused atleast in part by the increase in size in the S1'subsite. However, since Leu and Phe are notthat different in size and shape, it is likely thatmutational effects also reflect changes in thequality of the S1'-P1' interactions. Consideringthe similarity between Leu and Phe, the changesin substrate preferences that were obtained inthe present study are remarakbly large,especially for the dipeptide substrates. Thesechanges were obtained by considerableincreases in activity for substrates with a P1'Phe, and not primarily by deterioration ofactivity towards substrates with a P1' Leu (incontrast to (186). It is interesting to note that the16-fold increase in activity that the Leu202Tyrmutant displays towards FaGFa is one of thehighest found to date for a single mutant insimilar studies.

AcknowledgementsThe authors wish to thank Dr Theo

Sonke from DSM-Research for supplyingpUBTZ2 and helpful discussions.

Surface charge mutations

51

The effects of modifying the surface charge on thecatalytic activity of a thermolysin-like protease.

Abstract.The impact of long-range electrostatic

interactions on catalysis in the thermolysin-likeprotease from Bacillus stearothermophilus wasstudied by analysing the effects of inserting orremoving charges on the protein surface.Various mutations were introduced at sixdifferent positions and double-mutant cycleanalysis was used to study the extent to whichmutational effects were interdependent. Theeffects of single point mutations on the kcat/Km

were non-additive, even in cases where thepoint mutations were located 10 Å or more fromthe active site Zn2+ and separated from eachother by up to 25 Å. This shows that catalysis isaffected by large electrostatic networks thatinvolve major parts of the enzyme. Theinterdependence of mutations at positions asmuch as 25 Å apart in space also indicates thatother effects, such as active site dynamics, playan important role in determining active siteelectrostatics. Several mutations yielded asignificant increase in the activity, the mostactive (quadruple) mutant being almost fourtimes as active as the wild-type. In some casesthe shape of the pH-activity profile was changedsignificantly. Remarkably, large changes in thepH-optimum were not observed.

Introduction.The acceleration of reaction rates by

enzymes is one of the essential prerequisites forlife as we know it, and the multitude anddiversity of enzymes shows that it should bepossible to design an enzyme that will catalysealmost any reaction under almost any set ofconditions. To achieve a high rate-acceleration,enzymes rely on charged groups in their activesite that stabilise the transition state and

function as acid and base catalysts in thereaction. The kinetic parameters of enzymestherefore display a significant pH-dependence,which is determined by the pKa values of theactive site groups.

Since catalysis depends on intricateelectrostatic interactions, which may benoticeable over relatively large distances, ascompared to other short ranged interactionssuch as H-bonds and hydrophobic interactions,larger parts of an enzyme may be involved inoptimizing its catalytic centre than previouslythought. The long-range character ofelectrostatic effects is illustrated by a, verylimited, number of examples in the literature,showing that changes in surface charge atlocations as far as 15Å from a catalytic centremay affect enzyme activity (74, 191).Unfortunately, electrostatic interactions are hardto handle theoretically, not only because of theirlong-range character, but also because ofintrinsic theoretical difficulties. For example,most electrostatic models still use a single rigidprotein structure, and at most two dielectricconstants to account for all the dynamics of theprotein. This clearly is an oversimplification ofreality (192).

We have studied the contribution of longrange electrostatic interactions to catalysis byanalyzing the effects of a series of charge-mutations scattered over a larger part of thesurface of a thermolysin-like protease fromBacillus stearothermophilus (TLP-ste).Thermolysin-like proteases (TLPs) are membersof the peptidase family M4 (51) of whichthermolysin (TLN, EC 3.4.24.27) is theprototype. One of their characteristics is a zincion bound in the catalytic centre. The aminoacid sequences of several TLPs have been

Chapter 5

52

determined [see (51), or the Merops data base athttp://www.merops.co.uk/merops/famcards/m4.htm], and the three-dimensional structure ofTLPs isolated from several bacteria have beensolved i.e. those from TLPs from Bacillusthermoproteolyticus (52), Bacillus cereus (54),Pseudomonas aeruginosa (55) andStaphylococcus aureus (127). TLPs consist ofan α-helical C-terminal domain and a β-rich N-terminal domain. These two domains areconnected by a central α-helix, which is locatedat the bottom of the active site cleft and whichcontains several of the catalytically importantresidues. From X-ray structures of TLN-inhibitor complexes (52, 57-61), the active siteresidues have been identified and a mechanismhas been proposed (61, 62, 155). Recently, analternative mechanism has been proposed whichhas gained some support (63, 64). In bothproposed mechanisms residues Glu143, His231,Tyr157 and a Zn2+ bound water play importantroles during catalysis.

Mutations were introduced at six surfacepositions in TLP-ste, located at 10 - 15 Å fromthe catalytic centre. The single and multiplemutants that were obtained displayed varyingeffects on catalytic efficiency, includingconsiderable increases in activity. Double-mutant cycle analysis (193) was used to studythe additivity of mutational effects, whichshowed a remarkable interdependence of themutated residues. The results provide insight inthe complexity of predicting and interpretingelectrostatic effects in catalysis.

Materials and Methods

Modelling and mutant design.A three-dimensional model of TLP-ste

was built with WHAT-IF (140) using thecrystal structure of thermolysin as template, asdescribed elsewhere (78). The high sequenceidentity between thermolysin and TLP-ste(86%) indicates that the TLP-ste model is

sufficiently reliable for prediction and analysisof the effects of most amino acid substitutions(78, 141). Indeed, the TLP-ste model has beenused successfully for the design of variousstabilising mutations (43, 82, 177). Throughoutthis paper, residues are numbered according tothe corresponding residues in thermolysin.

All Glu, Gln, Asp and Asn residues thatare close to but not in the active site cleft andare positioned at the surface of the protein wereselected for mutagenesis. Position-specificrotamer searches (180) for the residues to beintroduced showed that all new side chainscould adopt a favourable rotamer withoutconcomitant introduction of steric overlap.

Molecular Biology.The nprT gene encoding the TLP of B.

stearothermophilus CU21 (136) (TLP-ste) wascloned, subcloned, and expressed as describedpreviously (178). Site-directed mutagenesis wasperformed on a subcloned fragment of the TLP-ste gene by the QuikChange site-directedmutagenesis kit from Stratagene, La Jolla, USA.The nucleotide sequences of mutated fragmentsof the nprT gene were verified by DNAsequencing and the mutated fragments weresubsequently cloned into the Bacillus expressionvector pGE501 (178) containing the TLP-stegene with a deletion of the previously subclonedfragment.

Production and Characterisation of MutantEnzymes.

Production and purification of theenzymes were performed as describedelsewhere (144). Prior to determining the kineticparameters, protease preparations were desaltedusing pre-packed PD-10 gel filtration columnssupplied by Amersham Pharmacia, Uppsala,Sweden.

Determination of kinetic constants.

Surface charge mutations

53

The kcat/Km values of the enzymes forthe furylacryloylated tripeptide 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine(FaGLA) obtained from Bachem AG,Bubendorf, Switzerland were determined at37°C, in a thermostated Perkin-Elmer Lambda11 spectrophotometer. The reaction mixture (1ml) contained 50 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris⋅HCl), 50mM 4-morpholineethanesulfonic acid (MES),pH 4.4 to 8.4 with 0.4 units interval, 5 mMCaCl2, 1% Me2SO, 1% 2-propanol, 0.01%Triton X-100 and 100 µM of substrate. Thereaction was followed by measuring thedecrease in absorption at 345 nm (∆ε345 = -317M-1⋅cm-1)(114). The stock solution of thefurylacryloylated tripeptide was prepared bydissolving the peptide in Me2SO. Apparentsecond order rate constants (kcat/Km) weredetermined by varying the enzymeconcentrations over a 50-fold range underpseudo-first-order conditions and measuring theinitial activity, essentially according to themethod described by Feder (114). All kcat/Km

values are the result of a linear regressionanalysis of at least 14 independentmeasurements. The error margins, defined bythe highest and lowest value of the 95%confidence interval of the linear regressionanalysis, were at most 15% of the values given.

Double-mutant cycle analysis.Determining the contribution of a single

amino acid to the activity or stability of aprotein by mutating that residue alone is oftenmisleading. This is because the neighbouringresidues often change their position slightly tocompensate for the mutation. A very illustrativerecent example of this effect is provided byAlbeck & Schreiber (194, 195) for the TEM-BLIP complex. To overcome this problem themethod of double-mutant cycle analysis hasbeen recommended (193, 196, 197).

Double-mutant cycle analysis uses theenergy difference between free enzyme plussubstrate and the enzyme-substrate complex inthe transition state (193, 196, 198). This energydifference, referred to as ∆G‡, reflects bindingenergy in the transition state and can be derivedfrom measured kcat/Km values using

∆G‡ = -RT ln (kcat/Km × h/kT).

Now, consider two residues A and B in theenzyme which do not interact. In that case theeffects of mutating these residues will beindependent and therefore additive. Expressedin the thermodynamic terms of Fig. 5.1:

∆∆G‡1 = ∆∆G‡

1' and ∆∆G‡2

= ∆∆G‡2'

∆∆G‡1 stands for the change in ∆G‡ upon

mutation A�A'. ∆∆G‡1 is calculated as follows

(using the annotation of Fig. 5.1):

∆∆G‡1=∆G‡

A'B-∆G‡AB

= -RT ln [(kcat/Km)A'B × h/kT ] + RT ln [(kcat/Km)AB × h/kT]

Figure 5.1. Double-mutant cycles. Residue A is mutatedto A' and residue B to B'. ∆G‡ is the energy differencebetween free enzyme plus substrate and the enzyme-substrate complex in the transition state. ∆∆G1

‡ is thechange in ∆G‡ upon mutating residue A, ∆∆G2

‡ is thechange in ∆G‡ upon mutating residue B. See text fordetails.

Chapter 5

54

∆∆G‡1= -RT ln [(kcat/Km)A'B/(kcat/Km)AB]

If the residues symbolized by A and B in Fig. 1somehow influence each other, the effect ofmutating one may become dependent onwhether or not the other is mutated too.Expressed in the terms of Fig. 1:

|∆∆G‡1 - ∆∆G‡

1'| = |∆∆G‡2 - ∆∆G‡

2'| ≠ 0

In which the term |∆∆G‡1 - ∆∆G‡

1'| is definedas the coupling energy. This term is zero if theeffects of the mutations are independent, but notso if they are dependent.

The experimental error in the calculated∆∆G‡ values is derived by inserting the highestand lowest values of the 95% confidenceinterval of the kcat/Km values in the equation for∆∆G‡. The experimental error of the couplingfactor is the sum of the errors in the ∆∆G‡

values. The coupling factor is significantly non-zero when the error is smaller than the value ofthe coupling factor.

Electrostatic calculations.The change in the electrostatic potential

at the Nδ1 of His231, the Oε1 of Gly143, and atthe oxygen of the water molecule bound to thecatalytic zinc ion was calculated using WHATIF (140) interfaced to DelPhi II (199). Adielectric constant of 4 was applied in theinterior of the protein and a dielectric constantof 80 was assigned to the solvent phase (200).The ionic strength in the calculations was set tomatch the experimental conditions at pH 7.0(210 mM). The ∆pKa can be calculated from theelectrostatic potential difference Φ using thefollowing formulas:

Φ=QG

and ∆G = -RT ln Ka

In which Q is the charge, Φ the electrostaticpotential energy in V, and G the free-energy.Rearangement of these formula's leads to anequation for the ∆pKa:

)10ln(RTpKa ∆Φ−=∆

Results.Mutant design and production of mutantproteins.

All mutants were constructed asdescribed in the Materials and Methods section.Fermentation and purification yields werenormal for all single and multiple mutants.Table V.I summarizes the characteristics of thesingle mutants, compared to the wild-type. Fig.5.2 gives an overview of the stereochemicalrelationship of the mutated surface residues toeach other and to the catalytically activeresidues and the Zn2+ . The closest contactdistance between the mutated residues and theactive site Zn2+ is generally between 10 and 15Å. The closest contact distance between themost remote mutated residues is approximately25 Å between residue 225 and residues 116 and119.

Characterisation of mutant proteases.To examine the pH-activity profiles of

the mutant enzymes, the kinetic parameters forthe reaction of wild-type and mutant TLP-steswith the tripeptide substrate FaGLA weredetermined at different pH-values. Fig. 5.3shows the pH-activity profile of the wild-typeTLP-ste and of all the single mutants that wereconstructed. The pH-activity profiles generallyshow minimal changes in the pH optimum, theonly exception being the relatively inactivemutant N227D, for which the profile shows asmall acidic shift. The profiles do show someconspicuous changes in shape, in particulararound the “second” pH optimum near pH 6,0.

Surface charge mutations

Charge effects.To analyze the possible electrostatic

effects on the active site, we calculatedmutational effects on the (calculated) pKa of

groups in the catalytic centre (Table V.I).Generally, the calculated effects on pKa’s weresmall and there is no clear overall correlationbetween the effects on activity and the effectson pKa’s in the active site. However, the

Table V.I. Characteristics of the TLP-ste variants.

Mutation kcat/Kma ∆G‡ ∆Charge ∆pKa

Glu143 Zn – H2O His231M-1⋅s-1 ×10-4 kJ⋅mol-1

Wild-type 6.1 47.6N116D 10.4 46.3 -1 0.4 0.2 0.1Q119R 12.8 45.7 +1 -0.1 -0.1 -0.1D150N 2.4 50.0 +1 -0.5 -0.6 -0.2D150E 11.5 46.0 0 0.1 0.2 0.1D150Q 7.6 47.0 +1 -0.5 -0.5 -0.2D213E 0.8 53.0 0 -0.1 -0.1 0.1Q225E 6.3 47.5 -1 0.1 0.1 0.1Q225R 8.2 46.9 +1 -0.1 -0.2 -0.3N227D 1.4 51.5 -1 0.2 0.2 0.3

aMeasurements were performed at pH 6.8, experimental errors are less than 15% of thevalues given.

Figure 5.2. Stereo plot of the stereochemical relationship between the mutated residues and thecatalytically active residues. The active site Zn2+ (small cross at centre), and active site residues Glu143 andHis231 are indicated.

55

Chapter 5

56

vareDcoshlepKacpoobunpoonacop

Hef

saththreto

F

TD

igure 5.3. pH-dependent activity profiles of TLP-ste and the single surface charge mutants. Left panel; ○LP-ste, ●N227D, □ Q119R, ■ D150Q, ▲D150N, and ◊ D213E. Right panel; ○ TLP-ste, ● N116D, Δ150E, ♦ Q225R, and × Q225E. Experimental errors are less than 15% of the values given.

rious mutations at position 150 showed alatively clear correlation; comparison of150D (wild-type) with D150N andmparison of D150E with D150Q (Table V.I)ows that removal of charge at position 150ads to a relatively large decrease in active sitea's which is accompanied by a decrease in

tivity. Multiple mutations were also made atsition 225, but here no clear correlation isserved: although replacement of thecharged Q225 by either a negative (Glu) or asitive (Arg) amino acid had opposite effects the calculated pKa’s, the effects on the pH-tivity profile and on the activity at the pHtimum were marginal and almost identical.

ydrogen-bonds and loop flexibility; possiblefects on dynamics.

Residues 116 and 119 are located in theme surface loop and share a hydrogen bond ine wild-type enzyme. Interestingly, changinge interaction between residues 116 and 119 byplacement of either one of those residues leads an increase in activity, regardless of the net

change in charge. This suggests that the increasein activity is due to effects other thanelectrostatics. An appealing hypothesis is thatthe increased activity is due to increasedmobility of the surface loop.

The mutations at position 150 alsoindicate that other effects may play a role, inaddition to expected effects on the pKa's ofactive site groups. This is clearly seen in acomparison of the activities of D150N andD150Q at pH 6.8. Both mutations remove thecharge at this position and are expected to yieldnearly equal ∆pKa's. However, D150N displaysa 60% drop in activity, whereas D150Q shows a25% increase in activity. Repositioning thecharge at position 150 as in D150E resulted in a90% increase in activity at pH 6.8.

Taken together, these observations showthat other factors, such as changes in loopflexibility due to disrupted or changedhydrogen-bonds and relatively small changes inthe spatial localization of relevant charges,contribute to the observed mutational effects.

Surface charge mutations

Combining mutations.To examine whether more active

enzymes could be obtained and to study theadditivity of the mutational effects, double,triple, and quadruple mutants were constructed.Table V.II summarizes the characteristics of the

purified multiple mutants and Fig. 5.4 showspH-activity profiles. The most active mutantsdisplayed a 3.7-fold increase in activity at pH6.8. Like for the single mutants, changes in pHoptimum were marginal, but some multiplemutants displayed distinct changes in the shape

F

○▲E

Table V.II. Characteristics of multiple mutants.

Mutation kcat/Kma ∆G‡ ∆Charge ∆pKa

Glu143 Zn–H2O His231M-1⋅s-1 ×10-4 kJ⋅mol-1

Wild-type 6.1 47.6N116D+D150Q 7.2 47.2 0 -0.1 -0.3 -0.1N116D+Q225R 6.0 47.7 0 0.3 0.0 -0.2D150E+Q225R 21.9 44.3 +1 0.0 0.0 -0.2D150Q+Q225R 6.3 47.5 +1 -0.6 -0.7 -0.5N116D+Q119R+Q225R 21.9 44.3 +1 0.2 -0.1 -0.3N116D+Q119R+D150E+Q225R 12.8 45.7 +1 0.3 0.1 -0.2N116D+Q119R+D150Q+Q225R 22.2 44.3 +2 -0.3 -0.6 -0.5

aMeasurements were performed at pH 6.8, experimental errors are less than 15% of the values given.

igure 5.4. pH-dependent activity profiles of TLP-ste and the multiple surface charge mutants. Left panel;

TLP-ste, ● N116D+D150Q, ∆ N116D+Q225R, and ▲ D150Q+Q225R. Right panel; Δ D150E+Q225R,

N116D+Q119R+Q225R, ◊ N116D+Q119R+D150E+Q225R, and ♦ N116D+Q119R+D150Q+Q225R.

57

xperimental errors are less than 15% of the values given.

Chapter 5

5

of the pH-activity profile. The "second" pHoptimum at pH 6,0 that is observed in the wild-type enzyme, has completely disappeared insome of the most active mutants, in particular inthe triple mutant N116D+Q119R+Q225R andthe quadruple mutant N116D+Q119R+D150Q+Q225R. Since the double optimum must be aresult of the ionisation constants of the catalyticresidues, the disappearance of this doubleoptimum indicates a change in active siteelectrostatics. However, as noted above withrespect to the changes in activity is no clearcorrelation existed with either ∆pKa or ∆charge.Interestingly, the results indicate considerablenon-additivity of mutational effects. Forexample, addition of the Q225R mutation toD150E increases activity considerably, whereasthe Q225R mutation had only marginal effectswhen introduced into the wild-type enzyme.

Calculation of coupling energies.To determine the interdependence of the

residues mutated in this study, double-mutantcycle analyses were performed as described inthe Materials and Methods section. Fig. 5.5shows the double-mutant cycles which can beconstructed from the available data. The ∆∆G‡

values and the coupling factors, were calculatedfrom the kcat/Km values presented in Tables V.Iand V.II.

The coupling factor in Fig. 5.5Aindicates that the effects of mutations at position116 and 150 are dependent on each other. Theeffects of mutating positions 116 and 225 aredependent on each other, as indicated by theircoupling factor in Fig. 5.5B. Fig. 5.5C showsthe dependence of mutations at position 150 and225. From these three cycles it can be concludedthat the effects of mutations at the positions 116,150 and 225 are dependent on each other.

Since the contributions of residues 116,150 and 225 to the pH-activity profile aredependent on each other, demonstrating that the

Fvs∆zmM

igure 5.5. Double-mutant cycle analysis. The ∆∆G‡

alues indicated, were calculated from the kcat/Km valueshown in Tables I and II. The coupling factor |∆∆G‡

1-∆G‡

1'| is indicated in the centre of each cycle. A nonero coupling factor indicates that the effects ofutations are dependent on each other. See Materials andethods for further details.

8

effects of residue 119 are dependent on any

Surface charge mutations

59

combination of these residues, provides strongevidence that the effects of all four positions aredependent on each other. Fig. 5.5E indicatesthat the effect of mutating residue 119 is indeeddependent on the residues present at positions116 and 225. Therefore, the effects of mutating116, 119, 150 and 225 are all dependent on eachother. This is a remarkable observationconsidering the closest contact distance ofalmost 25 Å between some of the mutatedamino acids and the fact that the closest contactdistance of the individual amino acids to theactive site residues are all 10 to 15 Å..

Fig. 5.5F and 5.5G both show a double-mutant cycle to determine whether the effect ofmutating residue 150 depends on the presenceof the combined mutations N116D+Q119R+Q225R. Surprisingly, whereas the effect ofD150Q depends on the individual mutationsN116D (Fig. 5.5A) and on Q225R (Fig. 5.5C),Fig. 5.5G shows no significant dependence ofthe D150Q mutation on the presence of the116+119+225 combination. Even moresurprising, Fig. 5.5F, showing the dependenceof the effect of D150E on the 116+119+225combination, shows the largest coupling factor.These results indicate that even though theeffects of mutating certain positions aredependent on each other, some mutations mightappear to be independent of each other,depending on which combination of aminoacids is present.

DiscussionModification of the surface charge

should, according to electrostatic theory, lead toa change in the active site electrostatics (74).Therefore, the catalytic performance of anenzyme may be modified without interferingwith the structure of the active site. Here, wehave engineered a considerable increase inactivity by modifying surface charges in TLP-ste. The most active mutants wereapproximately four times more active than the

wild-type. It is noteworthy that this increase wasachieved by mutations which are all far from theactive site. The distances between thecatalytically important Zn2+ and the mutatedresidues varied from 10 to 15 Å.

This paper has provided severalindications that the effects of surface chargemutations on catalysis result from factors whichare independent of the charge changes per se. Ingeneral, with few exceptions, the observedchanges in pH-activity profile and activity donot correlate with the expected change in pKavalues. This suggests that other than ∆charge-induced ∆pKa effects, that is, effects that are notaccounted for in the software used forcalculating pKa values, are important.

A second indication for the occurrenceof other than charge effects comes from thestudies on additivity of mutational effects. Ashas been observed before (191), the effects ofsurface charge mutations are expected to beadditive (201). Accordingly, we observedincreased effects upon combination of severalmutations. However, the mutational effects werefar from additive, as shown by the double-mutant cycle analysis. This observation is notsurprising for residues 116 and 119, which sharea hydrogen-bond in the wild-type enzyme.However, it is highly surprising to find that allresidues mutated in this study affect each other,that is, they seem to interact over distances aslong as 25 Å. The fact that the interdependentresidues are so far apart excludes the possibilityof direct contacts and also makes it unlikely thatcharge effects alone account for the mutationaleffects.

The third indication of other than chargeeffects is the discrepancy between the observedeffects on the activity, and the observed effectson the pH optimum. If the change in activity isthe result of a change in active site electrostaticsthen a change in the pH-optimum would also beexpected. Although changes in the shape of thepH-profile were observed, the pH optimum

Chapter 5

60

itself was not changed in the most activeenzyme variants. These three examplesprobably indicate that other than electrostaticeffects play a role, as previously suggested byNielsen et al. (72).

A change in the active site dynamicscould be responsible for a large part of theobserved effects. This suggestion is supportedby earlier work in which replacing unchargedresidues by other uncharged residues resulted inchanges in activity and in the pH-activityprofile that were similar to, or larger than, thechanges observed after introducing or removingcharges (72). Alternatively, a complexelectrostatic network may exist on the surface ofthe enzyme which makes the effect of a surfacecharge mutation unpredictable.

The fact that residues 25 Å apart arecoupled has serious consequences for modellingand calculation of the effects of chargemutations, since it implies that the effect of anymutation on the surface of an enzyme isdependent on the rest of the surface residues.

The coupling also implies that larger parts of anenzyme are involved in optimizing its catalyticcentre. If this is true, then nature probablyoptimised considerably more than just the activesite of enzymes during evolution. The large sizeof enzymes could be explained by the need tobalance all the interactions on the surface of anenzyme, and their influence on the catalyticcentre.

Considerable increases in catalyticactivity can be obtained by modification of thesurface charge. The most active mutant obtainedwas almost four times as active than the wild-type TLP-ste. However, the non-additivity ofthe mutations and their small effect on the pH-optimum show that important contributions suchas active site dynamics and unknownelectrostatic network effects exist that are notincluded in current electrostatic models.Reliable models and predictions as to how tomodify the pH profile of an enzyme requiresbetter understanding of these contributions.

Summary and general discussion

61

Summary and general discussion

Protein engineering is amultidisciplinary approach to study theproperties of proteins, combiningcomputational, physical, biochemical andgenetic techniques. The use of proteinengineering has dramatically increased theknowledge of protein structure, folding andcatalysis and the underlying structure-functionrelations. In this thesis, protein engineeringtechniques were used to study the determinantsof substrate specificity, structural flexibilityneeded for catalysis, and activity ofthermolysin-like proteases (TLPs) produced bymembers of the bacterial genus Bacillus.

The TLP family includes enzymes froma number of Gram-positive bacteria, includingpathogens such as Legionella, Listeria,Clostridium, Staphylococcus, Pseudomonas andVibrio. The TLPs from these organismscontribute to their pathogenicity. Furthermore,the active site organization of TLPs exhibitssimilarity to those of a number of eukaryoticmetallopeptidases, in particular to members ofthe matrix metalloproteases (MMP’s). Theselatter enzymes have been shown to be involvedin a number of important processes in man,including the processing of precursors that playmodulatory roles in the formation of tumors.

Several members of the TLP family areapplied in industry, e.g. in baking, brewing andleather processing. For example, thermolysin isused for the synthesis of the artificial sweeteneraspartame. TLPs are not only interesting from amedical and commercial perspective. Theavailability of an impressive amount ofsequence, structural and kinetic data renders thisgroup of proteases an ideal subject for rationaldesign strategies.

Chapter 1, the introduction, deals withgeneral aspects of enzyme classification,

enzymatic catalysis, substrate specificity andactive site electrostatics. The presentedliterature shows that protein engineering is avaluable method to analyze these aspects.

In Chapter 2 a number of TLPs has, forthe first time, been characterized with anidentical substrate set and a uniform set of assayconditions. Characterization with peptidesubstrates, as well as HPLC analysis of β-caseindigests, showed that the TLP family is ahomogeneous family in terms of catalysis, eventhough there is a significant degree of aminoacid sequence variation. The results of thisstudy showed that differences in substratespecificity within the M4 family do not correlatewith overall sequence differences, but dependon a small number of identifiable amino acids.Indeed, molecular modeling, followed by sitedirected mutagenesis of one of the substratebinding pocket residues of the TLP of Bacillusstearothermophilus (TLP-ste) converted thecatalytic characteristics of this variant into thatof thermolysin.

Chapter 3 shows the importance ofconserved glycines in proposed hinge-bendingregions by analyzing the effects of Gly→Alamutations on catalytic activity. The apparentimportance of conserved glycine residues inproposed hinge-bending regions for TLPactivity supports the idea that hinge-bending isan essential part of catalysis. The active site ofTLPs is located at the bottom of a cleft betweenthe N-terminal and C-terminal domain.Crystallographic studies have shown that theactive site cleft is more closed in ligand-bindingTLPs than in ligand-free TLPs. Accordingly, ithas been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in“closure” and “opening” of the active site cleft.Two hinge regions have been proposed. One is

Chapter 6

62

located around a conserved glycine 78, thesecond involves residues 135 and 136. Theimportance of conserved glycine residues inthese hinge regions was studied by analyzingthe effects of Gly→Ala mutations on catalyticactivity. Eight such mutations were made inTLP-ste and their effects on activity towardscasein and various peptide substrates weredetermined. Indeed glycines at poitions 78, 135and 136 turned out to be important for catalyticactivity. The apparent importance of conservedglycine residues in proposed hinge-bendingregions for TLP activity supports the idea thathinge-bending is an essential part of catalysis.

In Chapter 4 the properties of the majorspecificity determining hydrophobic S1' pocketare discussed. In TLP-ste, the hydrophobic S1'subsite is mainly formed by Phe130, Phe133,Val139 and Leu202. The effects on substratespecificity of replacing Leu202 by smaller (Gly,Ala, Val) and larger (Phe, Tyr) hydrophobicresidues were studied. The results showed thatthe wild-type S1' pocket is optimal for bindingleucine side chains. Reduction of the size ofresidue 202 resulted in a higher efficiencytowards substrates with Phe in the P1' position.Rather unexpectedly, the Leu202Phe andLeu202Tyr mutations, which were expected todecrease the size of the S1' subsite, resulted in alarge increase in activity towards dipeptidesubstrates with Phe in the P1' position. This isprobably due to the fact that 202Phe and 202Tyradopt a second possible rotamer which opens upthe subsite compared to Leu202 and whichfavours interactions with the substrate. Tovalidate these results variants of thermolysinwere constructed with changes in the S1'subsite. Thermolysin and TLP-ste variants withidentical S1' subsites were highly similar interms of their preference for Phe versus Leu inthe P1' position. The 16-fold increase in activityof the Leu202Tyr mutant towards a P1' Phecontaining substrate is one of the highest foundin the literature for a single mutant.

Chapter 5 describes the possibility ofchanging the active site electrostatics of TLP-steby inserting or removing charges on the proteinsurface by site-directed mutagenesis. Double-mutant cycle analysis was used to determinewhich of the mutated residues are independentof, and which depend on the amino acidspresent at the other positions chosen formutagenesis. The results showed that the effectson the kcat/Km of single point mutations werenon-additive, even in cases where the pointmutations were 10 Å or more from the activesite Zn2+ and separated from each other by up to25 Å. This lack of correlation with electrostatictheory implies that electrostatic networks areprobably more complex than previously thoughtand that other effects, such as active sitedynamics, may play an important role indetermining the active site electrostatics.Several mutations caused a significant increasein the activity, the most active mutant beingalmost four times as active as the wild-type. Theshape of the pH-activity profile was changedsignificantly. Remarkably, this was achievedwithout concomitant large changes of the pH-optimum of the enzyme.

In summary, the work described in thisthesis shows that the specificity of thermolysin-like proteases can be altered in a predictableway using protein engineering techniques. Thecomparison of the various wild-type proteases,in chapter 2, identified the substrate specificity-determining amino acids. The exploitation ofthis knowledge, in chapter 4, resulted in TLP-stevariants with distinctly altered specificityprofiles.

It is widely assumed that enzymesundergo hinge-bending motions duringcatalysis. However, only a few studies havebeen described in which hinge-bending wasanalyzed by rationally designed mutations. Theresults described in chapter 3 support the idea

Summary and general discussion

63

that hinge-bending is a critical feature ofcatalysis in TLPs.

Today, one of the biggest challenges isto rationally influence catalysis. The results inchapter 5 show that the catalysis of TLP-ste canbe changed by surface charge mutations.However, it is also clear that the results aredifficult to interpret with current electrostaticmodels of the active site. The surprisingobservation that many mutations on the surfaceof an enzyme can influence each other, eventhough they are far apart, leads to an interestingspeculation. The question; why are enzymes sobig maybe probably answered as follows: If anyresidue on the surface influences theelectrostatics of the active site, and if all thoseeffects are dependent on each other, then natureoptimized more than just the active site duringevolution.

In this thesis, the concept of proteinengineering, that is the multidisciplinary studyof structure-function relationships in proteins,has been succesfuly applied to elucidate some ofthe structure-function relationships thatdetermine the activity and specificity of TLPs.However, although the concept of proteinengineering has often been quite successful,many challenges remain.

Although the specificity determinants ofa number of hydrophobic binding pockets ofdifferent enzymes have been elucidated, manyattempts at rational design of a novel specificityhave met with unexpected problems. One suchproblem is the existence of previously unknownsecondary specificity determining elements.Another problem is that a convenient assay todetect enzymatic activity on an industrially ormedically relevant substrate can sometimes not

be developed. Therefore, model substrates areoften used during the engineering of a protease.However, these model substrates can haveconformations different from those on which theprotease acts. As a result, the obtained change inspecificity with model substrates could be non-existing on the natural substrate. A partialsolution to these problems is to carefully choosethe model substrates, such that they resemblethe substrate of interest as closely as possible.

Today the electrostatic determinants ofboth enzymatic activity and pH-optimum are aspoorly understood as the thermal stabilitydeterminants were fifteen years ago. However,the development of novel random mutagenesismethods and automated high throughputscreening should facilitate the elucidation of theelectrostatic determinants. The application ofthese random mutagenesis methods shouldquickly yield variant enzymes with alteredelectrostatic profiles. Analyzing these variantsby reverse engineering, that is, reverting themutant back to the wild-type step by step untilthe novel properties are lost, should yieldinsight into the electrostatic determinants. Inthis way, a substantial amount of knowledgecould probably be generated in less time than bythe conventional protein engineering approach.Subsequent application of protein engineeringtechniques might eventually lead to sufficientknowledge to rationally modify the activity andpH-optimum of enzymes.

The availability of a large number ofprimary sequences, various expression andpurification systems, a large body of kineticdata, and several tertiary structures solved by X-ray crystallography make TLPs an excellentmodel system to examine structure-functionrelationships by protein engineering.

Samenvatting en algemene discussie

65

Samenvatting en algemene discussie.

Een enzym is een eiwit dat eenchemische reactie katalyseert, het bestaat uit eenlange keten aminozuren. De eiwitketen is in hetalgemee op een complexe wijze gevouwen envormt zo een drie dimensionale structuur,vergelijkbaar met een kluwen wol. In de natuurkomen 20 verschillende aminozuren voor. Devolgorde van de aminozuren in combinatie metde manier waarop de eiwitketen is gevouwen,bepaalt de eigenschappen van een eiwit.

Protein engineering betekent ongeveer"eiwit bouwkunde". Protein engineering is eenmultidisciplinaire benadering om deeigenschappen van eiwitten te bestuderen.Protein engineering combineert theoretische,fysische, biochemische en genetische kennis entechnieken om eiwitten te bestuderen. Dezemultidisciplinaire aanpak heeft tot eendramatische toename van kennis geleid op hetgebied van eiwit structuur, eiwit vouwing enkatalyse en de daaraan ten grondslag liggendestructuur-functie principes.

In dit proefschrift zijn proteinengineering technieken gebruikt om de"thermolysine-achtige eiwit afbrekendeenzymen" (TLPs) te bestuderen. Deze enzymenworden geproduceerd door bacteriën van hetgenus Bacillus. Met name is aandacht besteedaan de achtergronden van de substraatspecificiteit, de structurele flexibiliteit die nodigis voor katalyse, en de activiteit van deze eiwitafbrekende enzymen. Eiwit afbrekendeenzymen worden ook wel proteasen genoemd.

Op het oppervlak van een proteasebevindt zich een "slot", dit is een inkeping waarhet af te breken eiwit in past. Het af te brekeneiwit is dan de "sleutel" en wordt het substraatgenoemd. Een protease kan dus alleen maareiwitten afbreken die in zijn slot passen. Voorieder aminozuur van het af te breken eiwit heefteen protease een inkeping, deze wordt substraat

bindings pocket genoemd. Aangezien eenprotease ook uit aminozuren bestaat, het isimmers zelf ook een eiwit, wordt een bindingspocket gevormd door een aantal aminozuren.Deze aminozuren bepalen de aard van depocket. De pocket kan water afstotend zijn(hydrofoob) of juist water lievend (hydrofiel),hij kan electrisch geladen zijn, of juist niet, hijkan groot zijn, of klein, hij kan electrostatischgepolariseerd zijn of niet. Al dezeeigenschappen zorgen ervoor dat het slot eenserie specifieke eigenschappen krijgt zodatalleen nog maar bepaalde sleutels passen. In eenwaterafstotend slot past geen waterlievendesleutel, in een klein slot past geen grote sleutel,enzovoort. Door het veranderen van het slot, datwil zeggen, door het veranderen van deaminozuren die de substraat bindende holtesvormen, zou dus de voorkeur voor een bepaaldsoort substraat kunnen veranderen.

De TLP familie omvat enzymen uit eengroot aantal Gram-positieve bacteriën,waaronder enkele pathogenen zoals Legionella,Listeria, Clostridium, Staphylococcus,Pseudomonas, en Vibrio. Aan deze pathogene,ofwel ziekte verwekkende bacteriën, dragen deTLPs bij tot de pathogeniciteit, het ziekteverwekkende vermogen van zulke bacteriën.Het actieve centrum van TLP enzymen, dat ishet deel van het enzym dat uiteindelijk het werkverricht, sterke overeenkomsten met enkeleeukaryote proteasen, in het bijzonder de matrixmetalloproteasen (MMPs). Deze MMPs op hunbeurt, blijken betrokken te zijn bij een aantalbelangrijke processen in de mens, waaronder,op indirecte wijze, de vorming van van tumoren.

Bacteriële TLPs worden toegepast in deindustrie, onder andere in bakkerijen,brouwerijen en in de leerlooierij. Thermolysine,de bekendste TLP, wordt gebruikt voor hetmaken van aspartaam, een veel gebruikt

Chapter 7

66

kunstmatige zoetmiddel. TLPs zijn echter nietalleen medisch en commercieel relevant. Doorde jaren heen zijn deze proteasen uitvoerigbestudeerd, hetgeen een grote hoeveelheid aaninformatie en kennis heeft opgeleverd. Dezegeaccumuleerde kennis maakt TLPs uitermategeschikt voor de multidisciplinaire proteinengineering aanpak ter doorgronding van destructuur-functie principes die ten grondslagliggen aan eiwit structuur en katalyse.

Hoofdstuk 1, de introductie, behandeltalgemene aspecten van enzym klassificatie,enzymatische catalyse, substraat specificiteit,thermostabiliteit, en de electrostatica van hetactieve centrum van proteasen, in het bijzondervan de TLPs. Uit de besproken literatuur blijktduidelijk dat protein engineering bij uitstek eenwaardevolle methode is om zulke aspecten tebestuderen.

In hoofdstuk 2 wordt voor het eerst eenaantal TLPs gekarakteriseerd met een identiekeset substraten onder uniforme omstandigheden.Karakterisatie met zowel dipeptide substratenals HPLC analyse van β-caseine afbraakproducten, toont aan dat de TLP familie,ondanks een grote variatie in aminozuursequentie, een in catalytisch opzicht homogenefamilie is. De resultaten van dit onderzoek latenduidelijk zien dat de verschillen in substraatspecificiteit niet samenhangen met algeheleverschillen in aminozuur volgorde, maar slechtsafhankelijk zijn van een beperkt aantalidentificeerbare aminozuren. Plaatsgerichtemutagenese van één van de substraatbindingsholte residuen van het TLP afkomstiguit Bacillus stearothermophilus (TLP-ste) leiddeer toe dat dit enzym de eigenschappen kreeg vanthermolysine, hetgeen er inderdaad op duidt datslechts een paar aminozuren van belang zijnwaar het gaat om de verschillen tussen denatuurlijk voorkomende TLPs.

In hoofstuk 3 wordt het belang vangeconserveerde glycines in de voorgesteldescharnier regio's aangetoond op grond van de

effecten van Gly→Ala mutaties op dekatalytische activiteit. Het aktieve centrum vande TLPs bevindt zich op de bodem van degroeve tussen het N-terminale en C-terminaledomein. Kristallografische studies hadden aleerder aangetoond dat de groeve meer geslotenis in TLPs die een substraat gebonden hebben,dan in TLPs zonder substraat. Op grond vandeze waarnemingen is verondersteld dat TLPseen scharnierbeweging vertonen gedurende hunkatalyse. Deze beweging resulteert in het openen sluiten van de katalytische groeve. Tweegebieden zijn voorgesteld als mogelijkescharnier gebieden. Eén is gelocaliseerd rond degeconserveerde glycine 78, bij het tweedegebied zijn residuen 135 en 136 betrokken. AchtGly�Ala mutaties werden geconstrueerd inTLP-ste en hun effect op de afbraak van caseineen verschillende kleine peptide substraten werdgeanalyseerd. Het bleek dat de glycine residuenop poisties 78, 135 en 136 inderdaad belangrijkzijn voor catalyse. Het duidelijke belang van degeconserveerde glycine residuen in devoorgestelde scharnier gebieden ondersteunt hetidee dat een scharnierbeweging een essentieelonderdeel is van de katalyse door TLPs.

In hoofdstuk 4 worden de eigenschappenbesproken van de voornaamste specificiteitsbepalende, hydrofobe bindingsholte S1'. De S1'holte wordt in TLP-ste voornamelijk gevormddoor Phe130, Phe133, Val139 en Leu202. In dehuidige studie werden de effecten onderzochtvan het vervangen van Leu202 door kleinere(Gly, Ala en Val) en door grotere (Phe en Tyr)hydrofobe aminozuren. De effecten van demutaties tonen aan dat de originele S1' holteoptimaal is voor de binding van leucinezijketens. Het reduceren van de grootte vanresidue 202 resulteerde in een hogere aktiviteitop substraten met een Phe op de P1' positie. DeLeu202Phe en de Leu202Tyr mutaties, waarvanwerd aangenomen dat zij de S1' holte zoudenverkleinen, zorgden voor een grote toename inde aktiviteit op substraten met een Phe op de P1'

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positie. Dit onverwachte effect wordtwaarschijnlijk veroorzaakt door deomstandigheid dat 202Phe en 202Tyr eenalternatieve rotameer adopteren die de holteverder opent dan een holte met Leu202. Dezeverder geopende holte bevordert interacties metsubstraten met een groot P1' aminozuur. Omdeze veronderstelling verder te onderbouwenwerden enkele varianten geconstrueerd vanthermolysine. Thermolysine en TLP-stevarianten die een identieke S1' holte bezitten,vertonen een kwalitatief gelijke voorkeur voorsubstraten met een P1' Phe versus substraten meteen P1' Leu. De 16-voudige toename van deactiviteit van de Leu202Tyr mutant ten opzichtevan het oorspronkelijke TLP-ste op substratenmet een P1' Phe is een van de sterkste aktiviteitstoenames die ooit voor een enkele mutatie iswaargenomen.

Hoofdstuk 5 beschrijft de mogelijkhedenom de electrostatische interacties van hetaktieve centrum van TLP-ste te wijzigen doorhet aanbrengen of verwijderen van electrischeladingen aan het oppervlak van het enzym.Dubbel-mutant cyclus analyse werd toegepastom te onderzoeken welke van de gemuteerderesiduën onafhankelijk, en welke afhankelijkvan elkaar waren. De resultaten tonen aan dat deeffecten op de katalytische efficientie (kcat/Km)van enkelvoudige puntmutaties niet additiefzijn, zelfs niet in die gevallen waarbij depuntmutaties 10 Å or meer van het zink ion inhet aktieve centrum en tot 25 Å van elkaarverwijderd zijn. Dit valt moeilijk te rijmen metde huidige electrostatische theorie en impliceertwaarschijnlijk dat electrostatische netwerkencomplexer zijn dan tot nu toe werdaangenomen. Verder impliceert het resultaat datandere dan electrostatische interactiesverantwoordelijk zijn voor voor een deel van dewaargenomen effecten. Hierbij kan gedachtworden aan de dynamica van het aktievecentrum. Verschillende mutaties leidden tot eensignificante toename van de aktiviteit. De meest

aktieve combinatiemutant was bijna vier maalaktiever dan het originele TLP-ste. De vorm vanhet pH-aktiviteits profiel veranderde significant.Opmerkelijk genoeg gebeurde dit zonder dat ereen grote verschuiving optrad in het pH-optimum.

Samenvattend kan men stellen dat het indit proefschrift gepresenteerde werk laat ziendat de specificiteit van thermolysine-achtigeproteasen op een voorspelbare wijze veranderdkan worden met behulp van protein engineeringtechnieken. De vergelijking van deverschillende wild-type proteasen, in hoofdstuk2, leidde tot de identificatie van aminozuren dieverantwoordelijk zijn voor de substraatspecificiteit. Door deze kennis aan te wenden,zoals beschreven in hoofdstuk 4, werd een TLP-ste variant geconstrueerd met een duidelijkveranderd specificiteitsprofiel.

Algemeen wordt aangenomen datenzymen scharnierbewegingen ondergaantijdens hun katalyse. Echter, tot nu toe zijn inslechts enkele studies rationeel ontworpenmutaties gebruikt om deze scharnierbeweging teonderzoeken. De resultaten van hoofdstuk 3ondersteunen het idee dat eenscharnierbeweging een fundamenteel onderdeelis van katalyse door TLPs.

Vandaag de dag is het op rationele wijzebeïnvloeden van katalyse een van de grootsteuitdagingen binnen de protein engineering. Deresultaten in hoofdstuk 5 laten zien dat dekatalyse van TLPs beïnvloed kan worden doorhet veranderen van de lading aan het oppervlakvan het enzym. Het is echter ook duidelijk datde resultaten moeilijk te interpreteren zijn metbehulp van de huidige electrostatische modellenvan het aktieve centrum. De verrassendeobservatie dat verschillende mutaties op hetoppervlak van een enzym, elkaar over groteafstand kunnen beïnvloeden, leidt totinteressante speculaties. De vraag waarom zijnenzymen zo groot kan waarschijnlijk als volgt

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beantwoord worden: Als ieder residue op hetoppervlak invloed uitoefent op de electrostaticavan het aktieve centrum, en als al dezeinvloeden onderling afhankelijk van elkaar zijn,dan heeft de natuur meer dan alleen het aktievecentrum geoptimaliseerd gedurende de evolutie.

Het protein engineering concept, demultidisciplinaire bestudering van structuur-functie relaties in eiwitten, is in dit proefschriftop succesvolle wijze toegepast om enkeleactiviteit en specificiteit bepalende structuur-functie relaties van TLPs op te helderen.Hoewel het protein engineering conceptregelmatig met succes is toegepast, bestaan ernog vele uitdagingen.

Ondanks de opheldering van despecificiteits determinanten van een aantalhydrofobe bindingsholten van verschillendeenzymen, worden pogingen om op rationelewijze een nieuwe substraat specificiteit teontwerpen dikwijls geconfronteerd metonverwachte problemen. Het bestaan vanvoorheen onbekende secundaire specificiteitsdeterminanten is hier een voorbeeld van. Eenander probleem is dat een eenvoudige test omde enzym activiteit op een industrieel ofmedisch relevant substraat te meten, soms nietkan worden ontwikkeld. Daarom wordt tijdenshet veranderen van een protease vaak gebruikgemaakt van model substraten. Deze modelsubstraten echter kunnen een andereconformatie hebben dan de substraten waarophet protease actief moet zijn. Het gevolg hiervankan zijn dat de verandering in specificiteit diemet model substraten kunnen wordenwaargenomen, zich niet voordoen metnatuurlijke substraten. Een gedeeltelijkeoplossing voor dit probleem is een zorgvuldigekeuze van de model substraten, zodat deze eenzo groot mogelijke overeenkomst vertonen methet natuurlijke substraat.

De electrostatische determinanten vanzowel de enzymatische activiteit als het pH-optimum zijn vandaag de dag net zo onduidelijkals de thermostabiliteits determinanten vijftienjaar geleden. Echter, de ontwikkeling vannieuwe random mutagenese methoden engeautomatiseerde methoden waarmee in kortetijd grote aantallen mutanten worden getest(high throughput screening) zouden deopheldering van de electrostatischedeterminanten kunnen vereenvoudigen. Detoepassing van de genoemde technieken kunnenin korte tijd nieuwe enzym varianten metveranderde electrostatische eigenschappenopleveren. Door deze varianten aan "reverseengineering" te onderwerpen, dat wil zeggen, zestap voor stap terug te veranderen naar het wild-type enzym totdat het enzym de nieuweeigenschappen heeft verloren, kan inzichtworden verkregen in de electrostatischedeterminanten. Op deze wijze kanwaarschijnlijk een substantiele hoeveelheidkennis worden vergaard in minder tijd dan viade conventionele protein engineering aanpak.De daarop volgende toepassing van proteinengineering technieken kan uiteindelijk leidentot voldoende kennis om op rationele wijze deactiviteit en het pH-optimum van enzymen teveranderen.

De beschikbaarheid van een groot aantalprimaire sequenties, verschillende expressie enzuiverings systemen, een grote hoeveelheidkinetische data, en enkele tertiaire structuren diezijn opgehelderd met behulp van röntgendiffractie maken van TLPs een uitstekend modelsysteem om structuur-functie relaties teonderzoeken met behulp van proteinengineering.

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List of publications

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List of Publications

de Kreij, A., van den Burg, B., Venema, G., Vriend, G., Eijsink, V.G.H., and Nielsen, J.E. (2001)The effects of modifying the surface charge on the catalytic activity of a thermolysin-like protease.(submitted)

de Kreij, A., van den Burg, B., Veltman, O.R., Vriend, G., Eijsink, V.G.H., and Venema, G. Theeffect of changing the hydrophobic S1' subsite of a thermolysin-like protease on substratespecificity (submitted)

de Kreij, A., Venema, G., and van den Burg, B. (2000) Substrate specificity in the highlyheterogeneous M4 peptidase family is determined by a small subset of amino acids. J.Biol.Chem.275, 31115-31120.

van den Burg, B., de Kreij, A., and Venema, G. (1999) Hydrolysis of industrial substrates by anextremely stable thermolysin-like protease variant obtained by protein engineering. BiotechnologyLetters 21, 537-542.

van den Burg, B., de Kreij, A., van der Veek, P., Mansfeld, J., and Venema, G. (1999)Characterization of a novel stable biocatalyst obtained by protein engineering. Biotechnology andapplied Biochemistry 30, 35-40.

Veltman, O.R., Eijsink, V.G.H., Vriend, G., de Kreij, A., Venema, G., and van den Burg, B. (1998)Probing catalytic hinge-bending motions in thermolysin-like proteases by glycine � alaninemutations. Biochemistry 37, 5305-5311.

Gilardi, G., de Kreij, A., Mei, G., Rosato, N., Finazzi-Agro, A. and Cass, A.E.G. (1995) Potentialof protein engineering for biosensor technology: a study on the maltose binding protein. ItalianBiochemical Society Transactions v.6, p.116.

Nawoord

77

NawoordEn toen was het af. Het verdwaasde gevoel na het proefschrift te hebben ingeleverd bij de

leescommissie is niet te beschrijven, maar al die mensen die mij voor zijn gegaan weten precieswaar ik het over heb. Hoewel die 8 flessen wijn die avond er misschien ook wel wat mee te makenhadden……

Als eerste wil ik mijn dank en waardering uitspreken voor de inzet van mijn beidepromotoren. Vincent en Gerard, jullie hebben samen het project geschreven en het geld losgekregen van NWO waardoor ik OIO kon worden. Zonder jullie coaching en inzet was dit werk niettot stand gekomen. Vincent, ik wil je nadrukkelijk bedanken voor het enorme geduld waarmee jijtelkens weer de volgende versie van een manuscript doornam, en de zeer nuttige en waardevolleideeën die je altijd aandroeg voor zowel experimenten als interpretatie. Je bent nooit te beroerdgeweest om mijn manuscripten te lezen, ook al ging dat soms kennelijk ten koste van je prive tijd,"ik heb het razend druk" is een bekende uitspraak. Gerard, de vituositeit waarmee jij mijn "te kortdoor de bocht" geschreven teksten wist te veranderen in wetenschappelijk verantwoord proza, is ietswat slechts weinigen jou na kunnen doen. Hoewel ik hier veel van heb geleerd betwijfel ik of ik hierooit zo bedreven in zal worden. In het rijtje hooggeleerde heren mag natuurlijk Gert Vriend nietontbreken. Gert, ik heb altijd genoten van jouw ongezouten, recht voor zijn raap aanpak. De sessiesin zowel Heidelberg als in Nijmegen zijn altijd zeer leerzaam en verhelderend geweest, en zonderjouw inzet was het neutrale protease project nooit zo ver gekomen. Zowel jij als Vincent zijn voormij twee belangrijke voorbeelden geweest van hoe een wetenschapper hoort te zijn; gepassioneerddoor zijn werk, gedreven om het hoogst haalbare er uit te halen, altijd enthousiast om samen metanderen te werken aan wetenschappelijke problemen, en bereid om persoonlijke offers te brengenom je doel te bereiken.

Waar er twee vechten, hebben er twee schuld. Bertus, terugkijkend op de tijd dat wij beidenin het lab rond hebben gelopen kan ik het alleen maar jammer vinden dat wij geen van beiden instaat zijn geweest om de kloof tussen ons te overbruggen. De samenwerking is helaas nooitoptimaal geweest, en dat heeft ons beide ongetwijfeld een aantal publicaties gekost. Ondanks dat,heb ook jij mij geleerd hoe je een publicatie op poten moet zetten. Het wild-type stuk had zonderjouw inzet nooit in JBC gestaan.

Rob, ook jou wil ik bedanken. Jij hebt mij in het begin uitstekend ingewijd in de traditiesvan de protein engineers. Binnenkort worden we door hetzelfde bedrijf betaald, maar helaas zittenwe dan toch nog zo'n 400km bij elkaar vandaan. Gelukkig kan je voortaan op zaterdag ochtendgewoon binnenlands bellen, dat scheelt met jouw gesprekstijd toch al gauw een mercedes per jaar.

Groningen was wel even wennen, maar ik heb de stad leren waarderen in de afgelopen vijfen half jaar. Het komt voor velen misschien als een verrassing, maar ik zal de stad wel missen als ikhier weg ben. Het is alleen zo jammer dat Groningen zo ver weg ligt van de rest van Nederland. Endan de rest van mijn collega's. Als eerste natuurlijk Arie, Mozes en Peter. Zonder jullie kwam ditlab krakend tot stilstand. Bedankt voor jullie inzet. Ik heb mij in het lab uitstekend vermaakt, enhoewel ik door mijn onderwerp misschien een buitenbeentje leek, verschilt mijn werk minder vandat van mijn collega's dan velen op het eerste gezicht denken. Het cultuur verschil tussen debacillen en lacto's is bijzonder kunstmatig, maar is altijd een vruchtbare voedingsbodem geweestvoor goede humor.

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Van al mijn vrienden wil ik in het bijzonder noemen Maarten, Chris, Leendert en Elise.Zonder jullie was het leven op het lab, maar ook daarbuiten, een saaie boel geweest. Jullie hebbeneen belangrijke plaats ingenomen in mijn tijd hier in Groningen, en we hebben veel lol gehad.Maarten, bedankt voor de gastvrijheid. Je warme brabantse inborst onderscheid je overduidelijk vande koude stugge noordelijke mentaliteit. Jouw computer kennis heeft vele problemen voor mijopgelost, en ervoor gezorgd dat ik geen digibeet meer ben. Chris, jongen, jij bent te goed voor dezewereld. Met je delft/leidse achtergrond zaten wij qua humor en aanpak duidelijk op dezelfdegolflengte. Jouw voorliefde voor dure electronica heeft de nodige mooie DVD avondjes opgeleverd,die ik bijzonder zal missen. Leendert, hang loose man! Alles wat ik over Vincent en Gert zei geldook voor jou. Ik hoop dat je snel een lab vind waar je wel op waarde wordt geschat. Elise, youstupid woman! Bedankt voor de sneak, het goede eten, en je gezelschap op de labzaal. Jewerkbesprekingen zijn de meest memorabele uit mijn tijd hier in Groningen, en duurden altijd tekort. Ik heb er alle vertrouwen in dat je ook zonder Maarten en mij veel lol kunt hebben in dit lab.

Als laatste wil ik mijn dank betuigen aan mijn ouders. Hoewel ik jullie meestal in hetongewisse heb gelaten over waar ik precies mee bezig was, en het voor jullie soms onbegrijpelijkmoet zijn geweest waarom ik de keuzes heb gemaakt die ik gemaakt heb, hebben jullie mij altijdgesteund. Jullie hulp en toeweiding is iets wat ik bijzonder waardeer, en zonder welke mijn leveneen stuk onaangenamer en moeilijker zou zijn geweest. Mijn dank voor alles.

Vrienden, bedankt!

StellingenBehorende bij het proefschrift

Engineering specificity and activity of thermolysin-like proteases from Bacillusvan Arno de Kreij

1. Het vervangen van een klein pocket residu door een groter pocket residu kan leiden totmeer ruimte voor een substraat in een bindings pocket (dit proefschrift, hoofdstuk 4).

2. De met behulp van random mutagenese vergaarde kennis zal er uiteindelijk voor zorgendat rational design succesvoller wordt dan random mutagenese gecombineerd metmultiscreening.

3. Zolang de R&D uitgaven van de nederlandse industrie ver achter blijven bij het OECDgemiddelde, is de nederlandse industrie geen serieuze gesprekspartner in de discussie overhet besteden van universitaire onderzoeksgelden.

4. Prozac, EPO, viagra, XTC en nandrolon; het neerzetten van een topprestatie is verwordentot het kiezen van het juiste preparaat voor het juiste moment.

5. Het overschrijden van de promotieduur is voor een belangrijk deel te wijten aan falendmanagement.

6. Je verdient pas het predicaat "people manager" als je ook overweg kunt met mensen diegeen vrienden van je zijn.

7. Zonder conflict geen vooruitgang.8. Genetica bedrijven aan een organisme waarvan het genoom niet bekend is, is achterhaald.9. Grootschalige investering in de Palestijnse economie, ook door de Europese

Gemeenschap, is de enige methode om aan de voortdurende crisis rond Israel en Palestinaeen einde te maken.

10. Greenpeace is een organisatie die op macht en invloed uit is, en daarbij de leugen nietschuwt.

11. De fixatie van Hollywood op een goede afloop, leidt regelmatig tot een slecht einde vanamerikaanse films.