PROBING THE TOPOGRAPHY OF THE ACTIVE SITEOF a-CHYMOTRYPSIN

BY BERNARD F. ERLANGER

DEPARTMENT OF MICROBIOLOGY, COLLEGE OF PHYSICIANS AND SURGEONS,

COLUMBIA UNIVERSITY

Communicated by Erwin Chargaff, May 16, 1967

This paper is a report of a study of the topography of the active center of a-chymotrypsin. In particular, it will deal with that portion involved in sub-strate binding and with its position in space relative to the nucleophilic serineresidue that is believed to function in the catalytic mechanism.1

Since an enzyme must accommodate a substrate, the simplest approach to anunderstanding of the topography of the former might be a study of the conformationof the latter. However, this is not feasible with chymotrypsin. A typical sub-strate of chymotrypsin is acetyl L-phenylalanine ethyl ester.1 There is much evi-dence linking the susceptibility of this substrate to the aromatic ring of the aminoacid (though aromaticity is not necessary since cyclic hexahydro derivatives arealso substrates)2 and to the acylamido portion of the molecule. Benzoylamidomay substitute for acetylamido and, in fact, yields a somewhat better substrate(higher keat)3 in the case of acyl L-tyrosine amide. However, since all of the abovesubstrates are flexible molecules and therefore able to assume many conformations,there is no way of knowing their conformation when bound to the active center ofa-chymotrypsin.In our early studies we chose to use an approach based upon the experiments

of Wilson with acetylcholinesterase.4 Acetylcholinesterase and chymotrypsinbelong to the class of enzymes called serine esterases.1 All members of this classcontain an unusually reactive serine residue which acts as a nucleophile in thecatalytic mechanism. They are inactivated by certain organophosphorus com-pounds by a reaction that can be understood within the context of the enzymaticmechanism of the serine esterases. Briefly, they participate in the acylation stepof the mechanism but deacylate at an extremely slow rate unless nucleophilesconsiderably stronger than water are introduced, e.g., hydroxylamine, oximes, orhydroxamic acids.

Wilson4 had shown that the specificity sites of diethylphosphoryl (DEP)-cholin-esterase were still available for binding of substrate-like molecules. It was possiblefor him to design reagents which were bound by the inactivated (phosphorylated)enzyme in such a way as to position their nucleophilic functional group one bondlength away from the phosphorus atom, thus ideally situated for a nucleophilicdisplacement reaction. From a consideration of the structure of his best reactiva-tors, he was able to map out some details of the topography of the active centerof acetylcholinesterase.

In our early experiments,' we utilized a similar approach with DEP-chymotryp-sin. A large number of oximes and hydroxamic acids were tested as reactivators.Two of the compounds were found to be unusually reactive: N-phenylbenzohydrox-amic acid and N-phenylnicotinohydroxamic acid. They were, respectively, 15and 8 times faster reactivators than either benzohydroxamic acid or N-phenyl-

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acetohydroxamic acid. Hence, it was tentatively concluded that DEP-chymotryp-sin had two sites capable of binding aromatic rings and that when it did so withN-phenylbenzohydroxamic acid and N-phenylnicotinohydroxamic acid, the nucleo-philic hydroxamic acid group was favorably situated for a displacement of thephosphate group.

Supporting evidence was obtained from experiments with DEP-trypsin.6 Sincethe specificity of trypsin is entirely different from that of chymotrypsin, N-phenyl-benzohydroxamic acid would not be expected to be more effective than benzo-hydroxamic acid or aliphatic hydroxamic acids. It was, in fact, somewhat lessactive.

Thus, our early experiments allowed us to conclude that the active center of a-chymotrypsin contained two ring structure-binding sites that could be utilizedby the enzyme in its catalytic mechanism. However, their positions in spacerelative to each other and to the active serine residue could not be deduced becauseof the structural flexibility of the reactivator molecules.

Later findings enabled us to define the topography more exactly. In 1963,it was found that diphenylcarbamyl chloride (DPCC) was a specific inactivatorof chymotrypsin.7 Like the organophosphates, it inactivated the enzyme by aprocess that made use of the catalytic mechanism of the latter. Subsequentstudies by Metzger and Wilson8 showed that methyl phenylcarbamyl chloridewas also an inactivator of a-chymotrypsin but that it was about 150 times lessactive than DPCC, indicating again the presence on the enzyme of two ring-binding sites which could orient the carbonyl carbon properly with respect to thereactive serine.

Construction of a space-filling model of DPCC revealed considerable stericinterference with the freedom of rotation of the two aromatic rings, but still enoughfreedom remained to make an unambiguous mapping of the active center unfeasi-ble.Encouraged by these findings, we set about to design a completely constrained

inactivator. Among the compounds synthesized was phenothiazine-N-carbonylchloride (PCC),9 the structure of which is given in Figure 1, along with several otherreagents specific for chymotrypsin. Its resemblance to DPCC is apparent; however,the additional sulfur bridge restricts the movement of the two aromatic ringscompletely. Even rotation around the N-C bond is restricted severely by neigh-boring hydrogens on the beuzene rings. Hence, fixing onie aromatic ring determinesthe 1)ositioIl in space of the susceptible carbonyl chloride bond. iPCC is an effectiveand specific inactivator of chymotrypsin. The second-order rate constant for itsinactivation of a-chymotrypsin (105 liter mole-' sec-) is about one fourth thatof DPCC. However, as shown by its reaction with OH-, it is inherently lessreactive than DPCC by a factor of about 20. Correction for this lower reactivityin nucleophilic reactions leads to the conclusion that lPCC is even a more specificreagent for a-chymotrypsin than is DPCC. Another interesting facet of thereaction of PCC with a-chymotrypsin is that spontaneous reactivation occurs at a

slow but measurable rate (k4 = 25.3 X 10-4 min' at 370, pH 7.55). PCC,therefore, is more like a susceptible substrate. This would imply that less distor-tion of the active center occurs as a result of acylation of the enzyme by PCC thanby DPCC.

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Cl Cl

C=O C=O

Diphenylcorbamyl Phenothiazine -N- CorbonylChloride Chloride

0 A

tteC5OCHI H C-OCH3Il"?H NH 0

0 iIi 0-D-i-Keto-3-Corbomethoxy- o

l,2,3,4,-Tetrchydroieoquinoline IfOH Benzoyl L-PhenylalanineC1. .,HMethyl Ester

N -PhenylbenzohydroxomicAcid

FIG. 1.-Chemrical structures of specific substrates, inactivators,and reactivators of chymotrypsin.

If we now construct models of all of the reagents shown in Figure 1, it becomespossible to map the topography of the active center of chymotrypsin. Amongthe reagents included is the substrate discovered by Hein et al.,'0 D-1-keto-3-carbomethoxy-1,2,3,4-tetrahydroisoquinoline (D-CDIC). D-CDIC can be lookedupon as a D-phenylalanine analogue of benzoyl L-phenylalanine methyl ester withone aromatic ring serving a dual role as the acylamido group and as the benzenering of phenylalanine. Surprisingly, the L-enantiomorph was a poor substrate.Wilson and ErlangerII provided an explanation for this unusual situation by postulat-ing that the aromatic ring was bound as if it were the benzoylamido group ofbenzoyl L-phenylalanine ethyl ester. This orientation will be utilized in thepresent discussion. An important aspect of this substrate is its highly constrainedstructure and hence its potential utility as a mapping tool.A model of benzoyl L-phenylalanine methyl ester was constructed in order to

find a conformation that could be assumed by all of the constrained reagents.Only one could be found; it is shown in Figure 2. The molecule on the upperleft is phenothiazine-N-carbonyl chloride; on the uppel right, beulzoyl L-phenyl-alanine methyl ester; on the lower right, D-CDIC. Asterisks indicate the targetcarbonyl carbons of the carboxylic acid derivatives; the leaving group, whetherchloride or ester, is marked X. As noted above, the D-substrate is arranged sothat its aromatic ring coincides with the benzoyl group (ring A) of benzoyl L-phenylalanine methyl ester. It should also be noted that the amide bonds coin-cide. The position of ring B could not be ascertained from models of benzoylL-phenylalanine methyl ester and the D-substrate alone. The rigidity of PCC,however, allows us to position ring B in space. Its plane is at about 900 to thatof ring A. The susceptible carbonyl carbons (asterisks) are attacked by theenzyme from below. As required, their positions in space coincide in the threemodels.

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FiG. 2.-Models of phenothiazine-N-carbonyl chlor- FIG. 3.-Models of N-phenyl-ide (upper left), benzoyl-Lphenylalanine methyl ester benzohydroxamic acid and benzoyl-(upper right), and D-l-keto-3-carbomethoxy-1,2,3,4- L-phenylalanine methyl ester (Xtetrahydroisoquinoline (lower right). Leaving group serving to indicate ester leavingindicated by X; acylamido oxygen, OI; carboxyl group). Arrow indicates carboxyloxygen, Oli. Enzyme makes attack from below on carbon which is in position thatcarboxyl carbon marked with asterisk. would be occupied by phosphorus

atom of inactivated chymotrypsin.Asterisk designates nucleophilicoxygen of hydroxamic acid. Otherdesignations similar to those inFig. 2.

In Figure 3 are models of benzoyl L-phenylalanine methyl ester (upper) and Nphenylbenzohydroxamic acid (lower). The benzene ring of benzohydroxamicacid is aligned with that of the benzoyl function of the substrate, bringing thecarbonyl groups in coincidence. Also, the N-phenyl moiety of the reactivator canbe practically superimposed upon the benzene ring of the phenylalanine residueof the substrate. An asterisk indicates the nucleophilic oxygen of the hydroxamicacid. It is nearer to ring A than is the target carbon atom of the substrate (arrow)and is below the plane of the latter, placing it in an ideal position for a nucleophilicattack on the carbonyl (or phosphoryl) group.'2

It is interesting to note that the reactivator can also be turned around so thatits N-phenyl group coincides with ring A as shown in Figure 4. Now the liucleo-philic oxygen falls in the same plane but one bond length to the right of the car-boxyl carbon-again properly placed. 13

Figure 5 is a drawing of what is believed to be the conformation of benzoyl L-phenylalanine methyl ester when it is bound to the enzyme. It is the same asshown with the space-filling models in the previous figures, but somewhat exploded(and distorted) to make its details more obvious. This conformation is assumedby the molecule as a result of interactions with three sites on the enzyme: twowhich bind rings A and B, respectively, and one which interacts with the amidefunction of the substrate. Acetyl-L-phenylalanine ethyl ester, which lacks ringA, assumes the reactive conformation by interaction of ring B and the amido group

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0 -' / en

A d C t

FIG. 4.-Same as Fig. 3, FIG. 5.-Sketch of conformation of benzoyl-Iphenylalanineexcept that positions of rings methyl ester when bound to surface of enzyme. E-OHA and B of N-phenylbenzohy- represents nucleophilic portion of enzyme making attack ondroxamic acid are interchanged. susceptible carboxyl carbon.

with the active center of the enzyme. This is sufficient for proper orientation ofthe susceptible carbethoxy function.The nucleophilic portion of the enzyme is represented by E-OH. It is attacking

0

the carbonyl in a direction perpendicular to the plane of the C-X group. Asnoted, the carboxyl carbon is 0.5 A above the plane of ring A and 1.2 A to the left ofplane B.We believe that this arrangement is in agreement with all of the experimental

data on chymotrypsin. In any case, it arises from our inactivation-reactivationstudies as well as from a consideration of the findings of others. There are severalimplications that follow from the suggested conformation:

(a) The carbomethoxy group of D-CDIC is equatorial when it is attacked bychymotrypsin. This is contrary to the suggestions of Hein and Niemann"4 andof Awad, Neurath, and Hartley,15 both of whom require that the carbomethoxygroup be axial to the ring. However, it is known16 that hydroxide ion preferentiallyhydrolyzes an equatorial carboxylic ester, and the recent work of Silver17 using4-t-butyl-cyclohexane substrates of chymotrypsin indicates that enzymic hydrolysisof the equatorial ester group is also favored over that of an axial ester. Thus, thepublished experimental evidence favors an equatorial conformation.

(b) The acylamido group plays an important part in the binding of benzoylL-phenylalanine methyl ester to the enzyme and is in the cis configuration. Thelatter is a requirement; it is not possible to construct the benzoyl L-phenylalaninemethyl ester model "properly" if a trans configuration is used. It is also likely,but not necessary for our hypothesis, that the amide function also plays an impor-

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taut part ill the hiitdliig of D)-CDIC; ald, of course, if' this case it, caI oily be illthe cis coillfiguratioiL'8 Silver'7 co(nsiders this to he the major defect of our modelaid, inl fact, lie dismisses it as a possibility for this otie reason, iniplying that flexiblesubstrates must have a trans amide bond inl solution. This leaves only the aromaticring as a participant in binding, a condition which, in our opinion, may not beadequate for the proper placement of the susceptible carbomethoxy group. Thereare a number of studies that have shown that amides can exist both in the cisand trans configurations in solution and can, in fact, be converted from one formto another rather easily. For example, Berger, Lowenstein, and M\eiboom'9have shown by N1IR studies that N,N-dimethylacetamide has a considerabledegree of free rotation around the amide bond in aqueous solution below pH 5.0.The same authors showed the same to be true for N-methylacetamide at pH'sbelow and above pH 5.0. They proposed that this free rotation occurred becauseof the existence of a small but significant quantity of a protonated, freely rotatingspecies in equilibrium with the unprotonated amide. Hanlon and Klotz20 haveshown that polyamino acids can be protonated at the peptide bond in solutionscontaining extremely low concentrations of trifluoroacetic acid and that the resultis an increase in free rotation around the peptide bond. Even formic acid hasbeen shown to be able to protonate peptide bonds.21 There is no reason to assumethat the same type of protonation, and hence free rotation, cannot take placeat the surface of the enzyme under the influence of properly placed proton donorsand that, subsequent to this, the cis conformation be favored by the geometry ofthe binding site. We believe that this is more likely than the suggestion that theacylamido groups are not important in the enzymic process.A recent paper by Cohen and Schultz22 also minimizes the importance of the

amido linkage of D-CDIC, and, like Silver,'7 suggests that it does not take part inthe binding interaction. The basis of their conclusion is that an ester analogue,D-methyl-3,4-dihydroisocoumarin-3-carboxylate, is an excellent substrate. Thisfinding, however, does not rule out the possibility that it is the carbonyl groupthat is essential for the binding reaction. In a paper of Silver and Sone,23 naphthoicacid nitrophenyl esters were shown to be substrates of chymotrypsin. A particu-larly good one was the 1,2-dihydronaphthoic acid derivative in which the estergroup was equatorial. Silver and Sone estimated that it was about as reactiveas the nitrophenyl ester of the D-CDIC would be. (The latter has never actuallybeen synthesized and studied.) This work is taken to support the minor importanceof the amido group in the reactivity of the D-substrate. There are two majorcriticisms of this work. First of all, no comparisons were made of the relativereactivities of the various esters in the absence of enzyme. Therefore, no accountwas taken of the steric and electronic factors that influence their behavior in nucleo-philic reactions. The second criticism concerns their extrapolation of the resultswith nitrophenyl esters to ethyl or methyl esters. It would be safer to wait untilstudies with the less active esters have been completed.

It is not a necessary condition of our hypothesis that the amide function play a

part in the binding and orientation of D-CDIC, although we think that it is a likelypossibility. It must, however, participate in the binding of flexible substrates,such as benzoyl L-phenylalanine ethyl ester. According to the models of Silver'7' 23and of Cohen and Schultz,22 it cannot participate in the binding of the D-CDIC

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because they require that the aromatic ring of the D-substrate be bound by thesite of the enzyme reserved for the benzene ring of phenylalanine (ring B of Fig. 4).This orientation of the substrate is the major difference betwveen their models andthe one described in. this paper. As far as we can determine using space-fillingmodels, their hypotheses cannot explain the activity of PCC.Our suggested conformation also agrees with the finding by Hein and Niemann24

that indole was a competitive inhibitor of the hydrolysis of the D-substrate butnot of the (slower) hydrolysis of its L-isomer, for which the inhibition was of themixed type. This can be explained if we assume that the L-isomer is hydrolyzedwhen bound to the other ring-binding site.25 In order to explain this phenomenon,Hein and Niemann postulated'4 that binding of the D-isomer occurred in a planethat subtended two binding sites rather than at either site. In a later paper,24 how-ever, the same authors stated that the experimental evidence suggested "that the pilocus is involved in binding of the (D) substrate." The P1 locus is their designationof the acyl binding site which in our case is defined as the site that binds the benzoylgroup (ringA). Their own model, however, could not be made to fit this conclusionand they were led to conclude that "the attempt to incorporate these conforma-tionally constrained molecules into the theory has done more to demonstrate thestriking differences between these compounds and the more conventional acylateda-amino acid derivatives than to clarify the theory." Cunningham' is critical ofthis conclusion and so are we.

Finally, if one measures the dimensions of the space-filling model of benzoyl L-phenylalanine methyl ester, the dimensions of the active site "cavity" of chymo-trypsin can be estimated. Excluding the space required for the leavinggroups,the dimensions of the cavity are 9.5X X11 X X 6.5 A, or approximately 680 A3.

In summary, the inactivation-reactivation studies on chymotrypsin have allowedus to propose a conformation assumed by susceptible substrates when bound to theenzyme. The proposed conformation has been shown to be in agreement withexperimental data originating in other laboratories as well as in our own. It re-mains now to test the suggested conformation by attempting to design new sub-strates of chymotrypsin.We wish to acknowledge the financial assistance of the Office of Naval Research, the National

Science Foundation, and the National Institutes of Health.

1 Cunningham, L. W., in Comprehensive Biochemistry, ed. M. Florkin and E. H. Stotz (NewYork: Elsevier Publishing Company, 1965), vol. 16, p. 85.

Jennings, R. R., and C. Niemann, J. Am. Chem. Soc., 75, 4687 (1953).'Green, N. M., and H. Neurath, in The Proteins, ed. H. Neurath and K. Bailey (New York:

Academic Press, 1954), vol. 2, pt. B, p. 1057.4Wilson, I. B., Federation Proc., 18, 752 (1959).

6Cohen, W., and B. F. Erlanger, J. Am. Chem. Soc., 82, 3928 (1960).Cohen, W., M. Lache, and B. F. Erlanger, Biochemistry, 1, 686 (1962).

7Erlanger, B. F., and W. Cohen, J. Am. Chem. Soc., 85, 348 (1963).8 Metzger, H. P., and I. B. Wilson, Biochemistry, 3, 926 (1964).Erlanger, B. F., S. M. Vratsanos, N. Wassermann, and A. G. Cooper, submitted for publica-

tion.10 Hein, G., R. B. McGriff, and C. Niemann, J. Am. Chem. Soc., 82, 1830 (1960).Wilson, I. B., and B. F. Erlanger, J. Am. Chem. Soc., 82, 6422 (1960).

12 The model of benzoylILphenylalanine methyl ester is used in Fig. 3 only to indicate the con-

formation that must be assumed by the reactivator. It is not intended to imply that N-phenyl-

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benzohydroxamic acid can displace an inactivator possessing the structure of the substrate. Infact, we have found that N-phenylbenzohydroxamic acid does not reactivate diphenylcarbamylchymotrypsin, presumably because the binding sites of the enzyme are already occupied by theinactivator and unavailable to the reactivator.

13 It is interesting to note at this point that N-phenylphenylacetohydroxamic acid has beenfound to be a specific reactivator of DEP-chymotrypsin (Cohen, W., and B. F. Erlanger, un-published experiments). Its reactivation rate is about one half that of N-phenylbenzohydroxamicacid, possibly because of the fact that its conformation is correct only when bound in a manneranalogous to that shown in Fig. 3. With its rings interchanged as in Fig. 4, its nucleophilicoxygen is displaced beyond the region occupied by the susceptible target group of the inactivatedenzyme.

14 Hein, G., and C. Niemann, these PROCEEDINGS, 47, 1341 (1961).15Awad, E. S., H. Neurath, and B. S. Hartley, J. Biol. Chem., 235, PC35 (1960).16 Gould, E. S., Mechanism and Structure in Organic Chemistry (New York: Holt, Rinehart

and Winston, Inc., 1959), p. 241.17 Silver, M. S., J. Am. Chem. Soc., 88, 4247 (1966).18 If we are correct in our assumption that all substrates of chymotrypsin when bound to the

enzyme have their acylamido groups in the cis configuration, it is a distinct advantage for the D-substrate to have an acylamido group already fixed in that manner. This may be one of thereasons for the fact that it is a good substrate. Furthermore, since native proteins contain peptidebonds stabilized in the trans configuration, we can readily explain why denatured proteins aremore rapidly hydrolyzed by enzymes: in the "random coil" the energy barrier between the cisand the trans configurations should be considerably lower than in the a-helix.

After this manuscript was completed, the author's attention was called to a report of the presenceof cis and trans peptide bonds in cyclo-(Gly-Phe-Leu-Gly-Phe-Leu) (D,L,L,L) and (L,L,L,L)(Bltha, K., J. Smol6kov4, and A. Vftek, Coll. Czech. Comm., 31, 4296 (1966)). The report wasbased on infrared studies.

19Berger, A., A. Lowenstein, and S. Meiboom, J. Am. Chem. Soc., 81, 62 (1959).20 Hanlon, S., and I. M. Klotz, Biochemistry, 4, 37 (1965).21 Klotz, I. M., S. F. Russo, Sue Hanlon, and M. A. Stake, J. Am. Chem. Soc., 86, 4774 (1964).22 Cohen, S. G., and R. M. Schultz, these PROCEEDINGS, 57, 243 (1967).23 Silver, M. S., and T. Sone, J. Am. Chem. Soc., 89, 457 (1967).24 Hein, G. E., and C. Niemann, J. Am. Chem. Soc., 84, 4487, 4495 (1962).25 Models of the L-isomer show that when bound in this way, it is necessary for the carbomethoxy

group to be axial to the amide ring in order to have it coincide with the position of the carbo-methoxy group of the D-substrate. This would explain, in part, why the L-isomer is a poorsubstrate.

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