transient intermediates in enzymology, 1964 –2008

Transient Intermediates inEnzymology, 1964 –2008

Published, JBC Papers in Press, March 9, 2015, DOI 10.1074/jbc.X115.650879

Perry Allen Frey

From the Department of Biochemistry, University of Wisconsin-Madison,Madison, Wisconsin 53706

Iwas born to John and Inez Frey in Plain City, Ohio, on November 14, 1935, and I receivedprimary and secondary education in the local public schools. My father worked in seasonallycomplementary trades as a sheep shearer and painting contractor. He hired me in the con-tracting business during school breaks through junior and senior high school and college. He

instilled in me the values of hard work and uncompromising honesty, which have served me well.My mother managed the local office of the General Telephone Company. I have been happilymarried to Carolyn Scott Frey since 1961. We have two daughters, Suzanne and Cynthia, and threegranddaughters, Samantha, Carrie, and Bonnie.

I served in the United States Army (1954 –1956), and I was educated in chemistry at The OhioState University (B.S., 1959). I was a chemist in the United States Public Health Service in 1960 –1963. I did graduate work at the University of Cincinnati Evening College until 1964 and then atthe University of Michigan and Brandeis University. I received my Ph.D. degree in biochemistryfrom Brandeis University under Robert H. Abeles in 1968. I was a postdoctoral fellow in chemistryat Harvard University under Frank H. Westheimer in 1968. Professor Abeles inspired me tobecome a biological chemist; he and Professor Westheimer trained me to address significantproblems by critical and imaginative methods.

I established my research program at The Ohio State University in 1969 as an assistant professorof chemistry and progressed to professor of chemistry in 1979. In 1981, I moved to the Universityof Wisconsin-Madison as a professor of biochemistry and the Institute for Enzyme Research. Iretired in 2008.

Beginning at the United States Public Health Service

Before pursuing my Ph.D. degree, I conducted laboratory research at the Sanitary EngineeringCenter of the United States Public Health Service in Cincinnati, Ohio, from 1960 to 1963. Thecenter later became part of the newly created Environmental Protection Agency. I worked as animmunochemist in the food chemistry laboratory. I purified the paralytic shellfish toxin, nowknown as saxitoxin, from mussels and linked it chemically to bovine serum albumin. I did notknow the chemical structure of saxitoxin at the time, although the elemental formula was avail-able. From this, it appeared that the molecule contained amine or guanidine groups. Applying thechemistry of the noted biochemist Heinz Fraenkel-Conrat, I used formaldehyde to create meth-ylene linkages between saxitoxin and serum albumin. The conjugate proved to be an immunogenin rabbits, allowing saxitoxin to serve as a hapten. The rabbits responded by producing anti-saxitoxin antibodies that neutralized the paralytic properties of saxitoxin. This work introducedme to immunology and toxicology, and the results led to the publication of my first research paper(1).

To gain more control over my professional life, I decided to move on to graduate school inpursuit of a Ph.D. degree. I joined Professor Abeles’ laboratory at the University of Michigan in

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 17, pp. 10610 –10626, April 24, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

10610 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 17 • APRIL 24, 2015

REFLECTIONS

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January 1964, and we moved to Brandeis University inAugust 1964. I did one year of postdoctoral research atHarvard in 1968 (Fig. 1).

Starting Research in Enzymology

I focused my enzymological research, first at The OhioState University in January 1969 and later at the Universityof Wisconsin-Madison in 1981, on the identification andchemical characterization of transient intermediates instepwise enzymatic reactions. Many enzymatic reactionsproceed in several chemical steps by way of transient inter-mediates. Often, the intermediates are high in energy andchemically unstable. A thorough understanding of anychemical process requires knowledge of the structures ofintermediates. Unraveling such reaction trajectoriesforms the basis of much chemical and biochemicalresearch. Moreover, according to Hammond’s postulate, ahigh-energy or transient intermediate must be regarded asclosely related to the transition state for a reaction. Theoryholds that enzyme catalysis arises from the stabilization oftransition states through tight binding. Therefore, anenzyme tightly binds a transition state-like intermediate.A stable molecule structurally similar to a transient inter-mediate must also be avidly bound by an enzyme. Thisexpectation forms the basis for the development of transi-tion state analogs as inhibitors for enzymes identified asdrug targets. For all of these reasons, the characterizationof transient intermediates should be regarded as an impor-tant objective in enzymology.

2-Acetylthiamin Diphosphate

In a central step of aerobic energy metabolism, pyruvateundergoes NAD�- and CoA-dependent dehydrogenationand decarboxylation to acetyl-CoA, NADH, and CO2. Thepyruvate dehydrogenase (PDH) complex catalyzes thisprocess at the active sites of three enzymes within thecomplex: pyruvate dehydrogenase (E1), dihydrolipoyl

acetyltransferase (E2), and dihydrolipoyl dehydrogenase(E3). E1 is thiamin diphosphate (ThDP)-dependent;

E2 has covalently bound lipoic acid (LipS2), and E3 is aflavoprotein with FAD. In the 1970s and 1980s, the overallreaction could be chemically described by Reactions 1–5,with the overall reaction being Reaction 6, the summationof Reactions 1–5.

Pyruvate � E1 � ThDP ¡ CO2 � E1 � hydroxyethyl-ThDP

E1 � hydroxyethyl-ThDP � LipS2-E2 º E1-ThDP

� acetyl-S-Lip(SH)-E2

Acetyl-S-Lip(SH)-E2 � CoASH º acetyl-CoA

� E2-Lip(SH)2

E2-Lip(SH)2 � E3-FAD º E2-LipS2 � dihydro-E3-FAD

Dihydro-E3-FAD � NAD� º E3-FAD � NADH

Pyruvate � CoASH � NAD� º Acetyl-CoA � CO2

� NADH

REACTIONS 1– 6

At the time, investigators addressed many questionsabout the PDH complexes, including the molecular weight,quaternary structure, spatial interactions among theenzymes, and regulation of activity. David Speckhard andMaria Maldonado in our group contributed to these issuesfor the PDH complex from Escherichia coli. AssociatesJohn Reardon and Heechung Yang synthesized alkylatingand acylating derivatives of undecagold complexes.Undecagold could be visualized and masses determined byscanning transmission electron microscopy. We preparedundecagold-labeled components of the PDH complex and,in collaborations with James Hainfeld and Joseph Wall atthe electron microscopy facility of the BrookhavenNational Laboratory, proceeded to clarify the molecularmasses of the core and reconstituted complexes (2–5). Wealso collaborated with my brother Professor Terrence G.Frey at San Diego State University to employ undecagoldclusters as labels of thiol groups in E2 and mitochondria.The undecagold-labeled dihydrolipoyl groups appeared aspairs of bright spots in microscope images (6).

My main chemical interest in the PDH complex was inReaction 2, the reductive transacetylation of lipoyl groupsin LipS2-E2 by E1�hydroxyethyl-ThDP. This reactioninvolves dehydrogenation of hydroxyethyl-ThDP coupledwith acetyl transfer to Lip(SH)2-E2 to form acetyl-S-Lip(SH)-E2. In NMR and kinetics experiments, Yuh-Shy-ong Yang in our group had shown S8-acetyllipoyl groups tobe produced in this reaction (7).

The mechanistic issue was whether 2-acetyl-ThDPcould be an intermediate in Reaction 2. Mechanisms not

FIGURE 1. Photo of the author, Perry Allen Frey.

REFLECTIONS: Transient Intermediates in Enzymology

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involving 2-acetyl-ThDP could be written, and no evidenceon this issue had been published. 2-Acetyl-ThDP hadnever been synthesized and was thought to undergohydrolysis too fast to be isolated and characterized. Ourgroup focused on the possible intermediacy of 2-acetyl-ThDP and obtained the results outlined in Fig. 2.

Although Reaction 1 was not strictly irreversible, rever-sal could not occur in the absence of CO2; exclusion of CO2made it irreversible. Because other steps were readilyreversible, Claire CaJacob and I considered whetherreversibility in those steps could lead to ThDP-dependenthydrolysis of acetyl-CoA through the intermediate forma-tion of 2-acetyl-ThDP in Reaction 2. Accordingly, incuba-tion of the PDH complex with NADH and acetyl-CoA inbuffer led to the ThDP-dependent hydrolysis of acetyl-CoA. This could be explained by the intermediate forma-tion of 2-acetyl-ThDP. Having no possibility to revert topyruvate in the absence of CO2, the system allowed2-acetyl-ThDP to undergo hydrolysis to acetate and ThDP(Fig. 2, right). Corazon Anonuevo Steginsky in our groupsimilarly observed ThDP-dependent hydrolysis of succi-nyl-CoA catalyzed by the analogous 2-ketoglutarate dehy-drogenase complex, presumably via the intermediate for-mation of 2-succinyl-ThDP (8, 9).

With Lim Leung and Douglas Flournoy, we also studiedthe reactions of E1 with 3-fluoropyruvate (Fig. 2, bottom).3-Fluoropyruvate reacted in place of pyruvate to undergoE1�ThDP-catalyzed decarboxylation according to Reaction1 to produce 2-fluoro-1-hydroxyethyl-ThDP. �,�-Elimi-nation of fluoride produced 2-enolacetyl-ThDP, which,upon ketonization, became 2-acetyl-ThDP. We then inves-tigated its further reactions within E1. We found thatE1�ThDP catalyzed the reaction of 3-fluoropyruvate toCO2, fluoride, and acetate (10). This suggested decarboxy-lation, fluoride elimination, and ketonization to 2-acetyl-ThDP, followed by hydrolysis. We then also found that3-fluoropyruvate, in the additional presence of dihydroli-poamide and CoASH with the PDH complex, reacted toform acetyl-CoA (11). Moreover, reaction of 3-fluoropyru-

vate with the PDH complex in the presence of dihydroli-poamide produced S-acetyldihydrolipoamide. All resultspointed to the intermediate formation of 2-acetyl-ThDPfrom 3-fluoropyruvate. No NAD� was required because3-fluoropyruvate was at the appropriate oxidation level toreact with dihydrolipoamide.

Our group took a more direct approach. Kenneth Gruyssynthesized 2-acetyl-ThDP by chromic acid oxidation ofsynthetic hydroxyethyl-ThDP (Fig. 2, left). ChristopherHalkides characterized 2-acetyl-ThDP in acidic aqueoussolutions by NMR as a mixture of three interconvertibleforms at equilibrium: the parent keto form, the ketohydrate, and the internal keto adduct with the 6-aminogroup of the pyrimidine ring (12). 2-Acetyl-ThDP provedto be stable in acidic solutions and prone to hydrolysis athigher pH values, displaying a unique pH-rate profile.

Brief reaction of [3-14C]pyruvate with the PDH complexin the presence of ThDP, NAD�, and CoASH followedclosely by an acid quench produced a 14C-labeled com-pound that co-migrated with authentic 2-acetyl-ThDPupon chromatographic separation (Fig. 2, top). Yields werecompatible with a transient intermediate, varying from 2 to12% in the amount of PDH complex, depending on condi-tions (12). The pH-rate profile for hydrolysis of 2-[14C]acetyl-ThDP from the trapping experiments provedto be identical to that of authentic synthetic 2-acetyl-ThDP, definitively identifying 2-[14C]acetyl-ThDP as atransient intermediate in the reaction of [3-14C]pyruvate.2-Acetyl-ThDP proved to be an intermediate in otherenzymatic reactions as well (13).

Galactose Metabolism in the Leloir Pathway

The Leloir pathway catalyzes the metabolism of galac-tosyl and glucosyl groups at the level of UDP-sugars.Galactokinase catalyzes the phosphorylation of galactoseto �-D-galactose-1-P; galactose-1-P uridylyltransferase(GalT) catalyzes the reaction of �-D-galactose-1-P withUDP-glucose to produce UDP-galactose and �-D-glucose-1-P. UDP-galactose 4-epimerase catalyzes the transforma-tion of UDP-galactose to UDP-glucose. The pathwaytransforms �-D-galactose into glucose-1-P for energymetabolism. The reversibility of 4-epimerase and uridylyl-transferase allows the production of UDP-galactose forgalactosyltransferases from UDP-glucose.

I initially addressed the reaction mechanism of theNAD�-dependent UDP-galactose 4-epimerase in E. coli.Up to that time, all NAD�-dependent hydride transfers inbiochemistry were strictly stereospecific, and 4-epimeraseraised the question of how non-stereospecificity couldoccur. The uridine diphosphoryl moiety of the substrateproved to play dual essential roles in the action of4-epimerase.

FIGURE 2. Overview of experiments identifying 2-acetyl-ThDP as anintermediate in reactions of the PDH complex.




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My associates Timple G. Wee, Shan Wong, and UanKang verified UDP-4-ketoglucose as a tightly bound inter-mediate and devised a clear understanding of the basis fornon-stereospecific action (reviewed in Ref. 14). The UDPmoiety proved to be the enzyme-binding anchor, with verylittle binding to the sugar. This allowed the glucosyl andgalactosyl moieties to undergo free rotations within theactive site. The constellation of glycosyl-C3(OH), C4(H),and C4(OH) proved to be similar in reactive rotamers ofgalactosyl and glucosyl moieties, so NAD� accessedC4(H) equally with either substrate.

In chemical, kinetic, and NMR spectroscopic experi-ments, my associates Yun-Hua Wong, George Flentke,Yijeng Liu, Janeen Vanhooke, Yaoming Wei, and JamesBurke showed that in a second essential role, binding ofthe UDP moiety chemically activated and increased thereduction potential of NAD� in 4-epimerase (15–19).Associates Adrian Hegeman, Barbara Gerratana, and Jef-frey Gross further showed how the NAD� and active siteresidues in the homologous dTDP-glucose 4,6-dehydra-tase catalyze dehydration of dTDP-glucose to dTDP-4-keto-6-deoxyglucose (20).

The overall 4-epimerase mechanism rationalizes hownature requires three enzymes to interconvert glucosyland galactosyl moieties differing only in stereochemicalconfiguration at C4. In nature, the installation of UDP asthe anchor in 4-epimerase requires phosphorylation ofgalactose by the kinase and uridylyl transfer by the trans-ferase (21). The Leloir pathway and 4-epimerase mecha-nism stand in contrast to the organic chemistry labora-tory, where NaBH4 readily reduces UDP-4-ketoglucose tothe mixture of UDP-galactose and UDP-glucose.

After mentioning to graduate student Lee-Jun Wongthat the galactosemia-defect enzyme, GalT, should func-tion in two steps through a uridylyl-enzyme intermediate,she verified ping-pong kinetics and isolated the covalentintermediate. Chemical degradation, site-directed mu-tagenesis and chemical rescue experiments in our labora-tory performed by Sue-Lein Yang, Teresa Field, Frank J.Ruzicka, and Jeongmin Kim identified His166 as the uri-dylylation site. Sandaruwan Geeganage proved the kineticcompetence of GalT His166-UMP. Details of chemicalmechanisms in the Leloir pathway are reviewed elsewhere(21).

Phosphoryl- and Nucleotidyl-enzymeIntermediates and Phosphorus Stereochemistry

My group’s focus on GalT broadened my attention tomechanisms of nucleotidyl- and phosphotransferases ingeneral. Nearly all were controversial regarding covalent

nucleotidyl- or phosphoryl-enzyme intermediates. Incases of ping-pong kinetics, covalent intermediates couldbe identified. In cases of sequential kinetics with ternary orquaternary complexes, the issue remained ambiguous.Often, covalent phosphoenzymes were reported, but thequestion of whether they represented artifacts remained.To address this issue, our group and those under FritzEckstein at the Max Planck Institute for ExperimentalMedicine, Jeremy R. Knowles at Harvard University, Ste-phen J. Benkovic at Pennsylvania State University,Wojciech Stec at the Polish Academy of Sciences, John A.Gerlt at the University of Illinois at Urbana-Champaign,and Gordon Lowe at the University of Oxford pursueddefinitive results through phosphorus stereochemistry.Biological phosphates, being achiral or prochiral at tetra-hedral phosphorus, had to be synthesized with P-substitu-ents such as S, 18O, and/or 17O stereospecifically placed tostudy stereochemistry (22).

The ping-pong mechanism of GalT implied overallretention of configuration at P� of UDP-glucose throughstereochemical inversion in two steps. Our laboratorydeveloped methods to synthesize P-chiral substrates forthis and a number of other enzymes. Stereochemical stud-ies also required methods to determine configuration atphosphorus in P-chiral substrates and products. Our lab-oratory developed 31P NMR and chemical/mass spectro-metric methods for assigning relative and, ultimately,absolute configurations at chiral phosphorus.

Two of our group’s first methods are illustrated in Fig. 3as examples. Kwan-Fu Sheu initially studied adenylatekinase, which catalyzes the phosphorylation of AMP byATP. He demonstrated first that the Rp- and Sp-epimers ofadenosine 5�-O-(1-thiodiphosphate) (ADP�S) displayeddifferent 31P NMR chemical shifts. This 31P NMR prop-erty subsequently could be employed to distinguishP-epimers and to assign P-configurations. He then showedthat adenylate kinase catalyzed stereospecific phosphory-lation of adenosine 5�-phosphorothioate to (Sp)-ADP�S(Fig. 3A) (23). Other phosphotransferases turned out alsoto display this property of stereospecific phosphorylationin our and other laboratories. This property enabled us toassign configurations to phosphorus in molecules withP-chiral [18O]phosphorothioate groups.

In stereochemical work on phosphotransferases, P-chiral adenosine 5�-O-(3-[3-18O]thiotriphosphate) ([�-18O]ATP�S) would be needed. In our group, John P.Richard and Hsu-Tso Ho synthesized molecules, such as(Sp)-[�-18O]ATP�S by the method outlined in Fig. 3B (24).This proved to be a substrate for most phosphotrans-ferases, and the configurations of the [18O]phosphoro-




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thioate groups in the products were determined by chem-ical, mass spectrometric, or 31P NMR methods. R. DouglasSammons, Thomas Meade, and Radha Iyengar devisedmethods for displacing sulfur from P-chiral phosphoro-thioate esters with 18O or 17O from water (25, 26). Ourgroup applied these methods to elucidate the stereochem-ical course of phosphotransfer (Table 1).

The outcomes of our stereochemical studies appear inTable 1. Reactions of P-chiral substrates in single-dis-placement mechanisms proceed with inversion of config-uration at phosphorus. Overall retention implicates a dou-ble-displacement mechanism. Accordingly, the reaction

of GalT proceeds with overall retention of configuration atP�. In an improbable scenario, overall retention couldresult from two retention steps. To test this remote possi-bility, Richard S. Brody and Abolfazl Arabshahi in ourgroup proved that the first step of the reaction, transfer ofthe uridylyl-�S group to His166, proceeds with inversion ofP�, proving overall retention in two inverting steps, asillustrated in Reactions 7 and 8 (27).

Phosphorus stereochemistry resolved all questions ofenzymatic single or double displacement at phosphorus.The results led also to the statement of the principle of“economy in the evolution of binding sites” in enzymes.

FIGURE 3. Syntheses of P-chiral biochemical phosphorothioates. A, adenylate kinase-catalyzed stereospecific phosphorylation of adenosine5�-phosphorothioate. B, chemical transformation of (Sp)-[�-18O2]ADP�S into (Rp)-[�-18O]ATP�S.

TABLE 1Stereochemistry of substitution at phosphorusApA, adenyl 3,5�-adenosine; OAA, oxalacetate; PEP, phosphoenolpyruvate.

Enzyme or chemical reaction Reaction Stereochemistry Ref.

Galactose-1-P uridylyltransferase UDP-Gal � Glc-1-Pº UDP-Glc � Gal-1-P Retention 27UDP-Glc � GalT His166ºGlc-1-P � GalT His166-UMP Inversion 28

UDP-glucose pyrophosphorylase UTP � Glc-1-Pº UDP-Glc � PPi Inversion 29Nucleoside-diphosphate kinase ATP � NDPºNTP � ADP Retention 27Adenylate kinase ATP � AMPº ADP � ADP Inversion 30Nucleoside phosphotransferase AMP � nucleosideºNMP � adenosine Retention 31Adenosine kinase ATP � adenosineº ADP � AMP Inversion 32Polynucleotide kinase ATP � 5�-HO-ApAº ADP � 5�-PO-ApA Inversion 33Glycerol kinase Glycerol � ATPº sn-glycerol-3-P Inversion 34Phosphoenolpyruvate carboxykinase

Mitochondrial OAA � GTP3 PEP � CO2 � GDP Inversion 35Cytosolic OAA � ITP3 PEP � CO2 � IDP Inversion 36

DNA polymerasePolymerase I Overall Inversion 37T7 polymerase Overall Inversion 38

Mevalonate-PP decarboxylaseMevalonate-PP � ATPº5-P-mevalonate-PP � ADP Inversion 39

Gentamicin nucleotidyltransferase Overall Inversion 40Adenylate cyclase (Bordetella pertussis) Overall Inversion 40FHIT (fragile histidine triad) Overall Retention 41Phosphoanhydride chemical synthesis Inversion 42Desulfurization of P-chiral ADP�S to P-chiral ��-18O�ADP Inversion 43Catalyzed thiophosphoanhydride synthesis Epimerization 44




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The first six entries in Table 1 exemplify the principle. In areversible nucleotidyl transfer or phosphotransfer, thereare two P-acceptors, one in the forward direction and theother in the reverse direction. When the P-acceptors arestructurally and electrostatically similar, the kinetic mech-anism is ping pong with overall retention at phosphorus.In these cases, the P-donor-binding site encompasses theP-acceptor subsite, with an intervening enzymatic nucleo-phile. Such a site is sufficient to support the overall reac-tion. The enzymatic nucleophile covalently mediatesphosphotransfer between the two P-acceptors, which bindto the P-acceptor subsite, but not at the same time, thusthe ping-pong mechanism and covalent E-P intermediate.Otherwise, when the P-acceptors are structurally andelectrostatically dissimilar, more binding sites are re-quired, the kinetics is sequential with ternary complexes,and the stereochemistry is inversion at phosphorus. In thelatter cases, there is no need for an enzymatic nucleophilewithin a ternary complex. This principle appears to hold inother classes of group transferases as well (45).

Identification and Characterization of RadicalIntermediates

In 1986, I refocused my research to the field of radicalenzymology, believing that much of the new chemistry inbiology might be radical chemistry. As a graduate studentin the 1960s, free radicals were regarded as off-limits forenzyme intermediates. Radicals were high-energy speciesand regarded as too highly reactive and difficult to controlto be accommodated within the active sites of enzymes. Bythe 1970s, a stable tyrosyl radical in the active site of E. coliribonucleotide reductase began to change things in enzy-mology. For some time, it was regarded as an anomaly andan exception. However, evidence gradually appeared indicat-ing the possibility of radicals in oxygenation reactions and inthe reactions of adenosylcobalamin, the vitamin B12 coen-zyme. I took an interest in S-adenosylmethionine, which Ithought might react as a radical. Ultimately, many newly dis-covered enzymes requiring S-adenosylmethionine have beenfound to function through radical mechanisms.

Fig. 4 shows the radicals 4a– 4n discovered by ourgroup. By purely chemical methods, we identified the rad-

icals shown in blue. For example, Ded-Shih Huang andFrank J. Ruzicka found isomerization of radicals 4a and 4bto be required, by precedent in organic chemistry, toexplain product formation in the action of methanemonooxygenase on 1,1-dimethylcyclopropane (46). Wecharacterized the other radicals shown in black in rapid-mixed freeze-quenched samples by EPR spectroscopy incollaboration with my Wisconsin colleague George H.Reed and his associates, who extracted the maximumstructural information from isotope-edited spectra.

Dioldehydrase and Ribonucleotide Reductase—My in-troduction to this field began with my training for aPh.D. degree under Professor Abeles. Dioldehydrase cat-alyzes the dehydration of propane-1,2-diol to propional-dehyde and proceeds by hydrogen transfer from C1 toC2. The reaction requires the coenzyme adenosylcobala-min, shown in Fig. 5A, in addition to dioldehydrase and apotassium ion.

Adenosylcobalamin-dependent isomerization reactionsfollow the pattern of Reaction 9, in which a group (R) ina substrate migrates from C� to C�, and a hydrogencross-migrates from C� to C�. The reaction of dioldehy-drase follows this pattern in the first step of Reaction 10,producing propionaldehyde hydrate. Dehydration leadsto propionaldehyde.

In my Ph.D. research on dioldehydrase, I found the fol-lowing results, among others. (i) Reaction of the complexof dioldehydrase and adenosylcobalamin (E�Cob(III)-CH2Ado) with the suicide inactivator glycolaldehydecleaved the Co–C5� bond to cob(II)alamin and 5�-deoxy-adenosine. (ii) Suicide inactivation by [2-3H]glycolalde-hyde produced 5�-[3H]deoxyadenosine. (iii) Reaction of[1-3H]propane-1,2-diol as the dioldehydrase substrateproduced [5�-3H]adenosylcobalamin. Reaction of [5�-3H]adenosylcobalamin with dioldehydrase and propane-1,2-diol produced [3H]propionaldehyde (47). Theseresults led directly to the conclusion that C5� in adeno-sylcobalamin mediates hydrogen transfer in reactionsof dioldehydrase. This function of adenosylcobalaminproved to hold for all other adenosylcobalamin-depend-ent enzymes (48).

REACTION 9

REACTION 10

REACTION 7

REACTION 8




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At the time, free radicals had not been discovered inenzymatic reactions and did not appear in my Ph.D. dis-sertation. Jack Halpern at The University of Chicago andRichard Finke at the University of Oregon later discov-ered the weakness of the cobalt– carbon bond in adeno-sylcobalamin and its tendency to undergo homolyticscission to cob(II)alamin and the 5�-deoxyadenosyl radi-cal (49, 50). This introduced radical chemistry into thefield of adenosylcobalamin, and radical intermediateswere found in adenosylcobalamin-dependent reactions.This led to the generic mechanism in Reactions 11–15for isomerizations,

E � Cob(III)-CH2Ado º E � Cob(II) � CH2Ado

E � Cob(II)-�CH2Ado � S–H º E � Cob(II)-CH3Ado-S�

E � Cob(II)-CH3Ado-S� º E � Cob(II)-CH3Ado-P�

E � Cob(II)-CH3Ado-P� º E � Cob(II)-�CH2Ado-P–H

E � Cob(II)-�CH2Ado-P–H º E � Cob(III)-CH2Ado � P–H

REACTIONS 11–15

where �CH2Ado is the 5�-deoxyadenosyl radical, S� is theradical arising from abstraction of a hydrogen from thesubstrate S–H, and P� is the product-related radical.

Thirty years after completing my Ph.D. research, Ireturned to dioldehydrase to complete analysis of suicideinactivation by glycolaldehyde (47). Associate AndreasAbend found that suicide inactivation led to radical 4c inFig. 4, the product of hydrogen abstraction from glycolal-dehyde (51, 52).

In our group, Philip A. Schwartz showed that photolysisof adenosylcobalamin in the presence of O2 produces5�-peroxyadenosine through the reaction of O2 with the5�-deoxyadenosyl radical (4e) in Fig. 4 (53). Schwartz fur-ther showed that O2-dependent cleavage of adenosylco-balamin at the active site of dioldehydrase also produces5�-peroxyadenosine through radical 4e (54).

Class II ribonucleotide reductase catalyzes the adeno-sylcobalamin-dependent reduction of ribonucleoside

FIGURE 4. Radical intermediates discovered in enzymatic active sites. Radicals 4a and 4b, methane monooxygenase; 4c, dioldehydrase; 4d,lysine 2,3-aminomutase and dioldehydrase; 4e, dioldehydrase and class II ribonucleotide reductase; 4f, glutamate 2,3-aminomutase; 4g– 4j, lysine2,3-aminomutase; and 4k– 4n, lysine 5,6-aminomutase.

FIGURE 5. Structures of adenosylcobalamin and complex of S-adenosylmethionine and [4Fe-4S].




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triphosphates to 2�-deoxynucleoside triphosphates by amechanism requiring the intermediate formation of a thiylradical at Cys408. Andreas Abend in our group synthesized5�-(R)-[5�-2H]adenosylcobalamin and demonstrated thatribonucleoside-triphosphate reductase catalyzes epimeri-zation of C5� in the presence of the allosteric activatordGTP. Dawei Chen in our group carried out a detailedkinetic analysis and proved that C5� epimerization pro-ceeds faster than reduction of the substrate. He furtherproved that the inactive mutant reductases C408A andC408S also catalyze C5� epimerization faster than theoverall reduction by wild-type reductase. The resultsimplicated the 5�-deoxyadenosyl radical (4e) as an inter-mediate in the generation of the Cys408 thiyl radical (55).JoAnne Stubbe at the Massachusetts Institute of Technol-ogy collaborated in this research.

Lysine 2,3-Aminomutase—In 1970, Horace A. Barkerand associates at the University of California, Berkeley,discovered clostridial lysine 2,3-aminomutase in bacte-rial lysine metabolism (56). The transformation of L-ly-sine into L-�-lysine, a 2,3-amino migration, followed thepattern of adenosylcobalamin-dependent enzymes (Re-action 9) but did not involve adenosylcobalamin. Instead,the enzyme required S-adenosylmethionine and pyri-doxal phosphate (PLP), and it contained a trace of iron.

Adenosylcobalamin and S-adenosylmethionine sharethe 5�-deoxyadenosyl moiety (Fig. 5, A and B) but areotherwise chemically different. In particular, the S�–C5�bond in S-adenosylmethionine is �30 kcal/mol strongerthan the Co–C5� bond in adenosylcobalamin.

Graduate student Marcia Moss and I began studies oflysine 2,3-aminomutase, and I continued with Janina Ba-raniak, Marcus Ballinger, Bin Song, Robert M. Petrovich,Kafryn Lieder, Weiming Wu, Elham Behshad, JenniferMiller, Dawei Chen, Frank J. Ruzicka, and Olafur Mag-nusson. The reaction of clostridial lysine 5,6-aminomu-tase, a 5,6-amino migration discovered by Thressa C.

Stadtman at the National Institutes of Health (57), alsofollowed the course of Reaction 9 and required adenosyl-cobalamin, not S-adenosylmethionine.

In our first work on lysine 2,3-aminomutase, Mossshowed that tritium in [5�-3H]adenosylmethionine ap-peared in L-lysine and L-�-lysine. This implicated the 5�-deoxyadenosyl moiety of S-adenosylmethionine in hy-drogen transfer, as in adenosylcobalamin-dependentreactions (58, 59). We wrote the radical mechanism inFig. 6 to account for our results.

Of the four radicals in Fig. 6, our group characterizedthree by rapid-mix/freeze-quench EPR and pulsed mag-netic resonance spectroscopy. The kinetically competentC2 radical in Fig. 6 appeared in the steady state of the re-action of lysine as radical 4g (Fig. 4) (60 – 63). The enan-tiomeric radical 4h appeared in the steady state in theaction of E. coli lysine 2,3-aminomutase (64). Senior as-sociate Frank J. Ruzicka discovered clostridial glutamate2,3-aminomutase and found intermediate radical 4f (65).

The other radicals in Fig. 6 are too high in energy to beobserved by EPR. In radicals 4f–4h, the unpaired elec-trons are stabilized by delocalization into the carboxylategroup. High-energy radicals in Fig. 6 can be stabilized byadjacent, sterically compact, functional groups that delo-calize the unpaired electron. 4-Thia-L-lysine reacts as asubstrate for lysine 2,3-aminomutase, and the substrate-related radical 4i appears in the steady state (66, 67).Trans-4,5-dehydro-L-lysine reacts as a suicide inactivatorof lysine 2,3-aminomutase and is converted to the allylicradical 4j, with cleavage of the S�–C5� bond and forma-tion of 5�-deoxyadenosine (68). Dehydration betweenC3� and C4� of the adenosyl moiety in S-adenosylme-thionine leads to S-3�,4�-anhydroadenosylmethionine,which functions as a coenzyme for lysine 2,3-aminomu-tase. In reaction with lysine 2,3-aminomutase and lysine,the EPR spectrum of 5�-deoxy-3�,4�-anhydroadenosine-5�-yl signals the transient appearance of the kinetically

FIGURE 6. Radical mechanism in the reaction of lysine 2,3-aminomutase.




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competent radical 4d (69). In the reaction of dioldehy-drase with the analogous 3�,4-anhydroadenosylcobala-min, radical 4d appears and interacts magnetically withcob(II)alamin in a triplet spin system (70).

Our group turned its attention to the theretofore un-known mechanism by which lysine 2,3-aminomutasepromotes cleavage of S-adenosylmethionine to the 5�-deoxyadenosyl radical. With graduate student Robert M.Petrovich, we found lysine 2,3-aminomutase to harbor acatalytically essential [4Fe-4S] cluster (71, 72). One ironproved to be unique in that it could be removed by con-trolled oxidation to form a [3Fe-4S] cluster and reduc-tively restored with FeSO4 and a reducing agent. In ad-dressing cleavage of the S�–C5� bond, Squire J. Bookersynthesized Se-adenosylselenomethionine and found itto activate lysine 2,3-aminomutase. We then collabo-rated with Robert Scott and Nathaniel Cosper at theUniversity of Georgia, experts in selenium x-ray absorp-tion spectroscopy, to determine the fate of seleniumupon cleavage of the Se�–C5� bond, with particular ref-erence to whether selenium would become a ligand tothe unique iron in [4Fe-4S]. Upon cleavage of Se-adeno-sylselenomethionine at the active site by reaction withtrans-4,5-dehydro-L-lysine to form the allylic radical 3j(Fig. 4), the x-ray absorption data revealed direct Fe–Seligation (73). We concluded that upon cleavage of Se�–C5�, selenium became ligated to the unique iron in thecluster. Subsequent work with Dawei Chen in a collabo-ration with Brian Hoffman and Charles Walsby atNorthwestern University showed that the interaction ofS-adenosylmethionine with the [4Fe-4S] cluster entaileddirect ligation of the methionyl amino and carboxylategroup with the unique iron (74). This interaction hadbeen found in the pyruvate formate-lyase activase, an-other S-adenosylmethionine enzyme (75). Integratingthe spectroscopic results, we wrote the mechanism inReaction 16 for the cleavage of S-adenosylmethionineinto the 5�-deoxyadenosyl radical (74).

Senior associate Frank J. Ruzicka obtained diffraction-quality crystals of lysine 2,3-aminomutase in complexwith S-adenosylmethionine and lysine in our laboratory.We collaborated with Professor Dagmar Ringe andBryan Lepore at Brandeis University to obtain a molecu-lar structure (76). The structure fully supported all that

had been found by spectroscopy and providing detailedinformation about the molecular structure and all lysine2,3-aminomutase contacts with Se-adenosylselenome-thionine, lysine, and PLP.

With associates Glen T. Hinckley and Susan C. Wang,we addressed the energetics of one-electron reductivecleavage of S-adenosylmethionine to the 5�-deoxyadeno-syl radical. The electrochemical reduction potential forthe [4Fe-4S] cluster in lysine 2,3-aminomutase proved tobe �0.43 V in the presence of S-adenosylmethionine(77). The reduction potential for a generic trialkyl sulfo-nium ion (�1.8 V) gave a difference of 1.4 V, meaningthat electron transfer would not be possible unless thegap in reduction potentials could be decreased. Electro-chemical data showed that the addition of a surrogate forlysine (alanine � ethylamine) lowered the reduction po-tential for [4Fe-4S] in lysine 2,3-aminomutase to �0.6 V.Moreover, the reduction potential for S-adenosylmethio-nine bound to [4Fe-4S] at the active site proved to be�0.99 V (78). Thus, binding of lysine and S-adenosylme-thionine closed the gap in reduction potentials from 1.4to 0.4 V, enabling one-electron transfer.

In 1984, Joachim Knappe at the University of Heidel-berg found the activation of the pyruvate formate-lyaseactivase by S-adenosylmethionine to be accompanied byglycyl radical formation in pyruvate formate-lyase andproduction of 5�-deoxyadenosine (79). Joan B. Broderick,now at Montana State University, and associates discov-ered the iron-sulfur cluster in pyruvate formate-lyase ac-tivase, and in the year 2000, they showed it to be [4Fe-4S] (80). The 1990s saw the discovery of biotin synthase,lipoyl synthase, and class III ribonucleotide reductase asS-adenosylmethionine-activated [4Fe-4S] enzymes (81–87, 90). All catalyzed chemically difficult reactions in-volving C–H cleavage. By the turn of the century, thegenes encoding these proteins and lysine 2,3-aminomu-tase had been cloned and sequenced. Heidi J. Sofia at theNational Human Genome Research Institute and associ-ates found the motif CxxxCxxC in common among theseenzymes. This motif proved to bind the [4Fe-4S] clus-ters. Sofia and colleagues searched the genomic databasefor proteins with this motif and found �600 proteins in2001 involved in highly diverse biological processes (88).She named them the radical SAM superfamily. By 2008, thesuperfamily had grown to �2800 proteins in over 40 fami-lies (89). The superfamily has grown to �48,000 proteins innearly 70 families (Structure-Function Linkage Database)that catalyze complex reactions, including repair of thyminedimers, vitamin biosynthesis, antibiotic biosynthesis, meth-ylation of DNA, anaerobic heme biosynthesis, carbide in-

REACTION 16




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sertion into the FeMo cofactor of nitrogenase, methano-genesis, and other chemically difficult reactions, oftenpresumably by radical mechanisms (91, 92).

Lysine 5,6-Aminomutase—The internal transfer of theC6 amino group to C5 of D-lysine, L-lysine, or L-�-lysine iscatalyzed by lysine 5,6-aminomutase, a PLP enzyme. 5,6-Aminomutase requires adenosylcobalamin but neitherS-adenosylmethionine nor iron (Fig. 5A). A chemical mech-anism analogous to that in Fig. 6 for lysine 2,3-aminomu-tase can be written, with substitution of adenosylcobalaminas the source of the 5�-deoxyadenosyl radical. In this mech-anism, the initial substrate radical would be the C5 lysyl-PLP radical, and the rearranged product-related radicalwould be the C6 methylene radical.

Graduate student Christopher H. Chang initiated re-search in our group on the 5,6-aminomutase (93). ElhamBehshad and Kuo-Hsiang Tang carried the work for-ward. No radicals could be detected by EPR spectroscopyin the steady states of reactions of any lysine substrate.This could be attributed to the high energy of all of theputative radicals that would appear in the mechanism.However, the lysine 2,3-aminomutase substrate 4-thia-L-lysine stabilized the C3 radical in Fig. 6, and the 4-thiagroup could be expected to stabilize equally well an un-paired electron at C5. Accordingly, Tang discovered rad-icals 4k– 4n in the reactions of the substrate analogs4-thia-D- and 4-thia-L-lysine (94). Radicals 4k and 4l inFig. 6 are analogs of C5 radical intermediates in the reac-tions of D- or L-lysine. The molecular structure of theresting enzyme with cobalamin and PLP bound, obtainedin collaboration with Catherine Drennan and FrederickBerkovitch at the Massachusetts Institute of Technology,implies a major structural reorganization attending thebinding of lysine (95).

In research on aminomutases, I expected to find S-ad-enosylmethionine to be an evolutionary predecessor ofthe more complex adenosylcobalamin, which is pro-duced in 24 biosynthetic steps. A corollary might be thatthe more elegant adenosylcobalamin dominates over the“inferior” predecessor. The results on lysine 2,3-amino-mutase support S-adenosylmethionine as a possiblepredecessor, and it is as active as any adenosylcobalaminenzyme. However, the sheer size of the radical SAM su-perfamily does not support adenosylcobalamin as a supe-rior radical initiator.

Role of Low-barrier Hydrogen Bonding in SerineProtease Reactions

Low-barrier Hydrogen Bonding in Chymotrypsin—I shallexplain how I came to study strong low-barrier hydrogen

bonds (LBHBs), with special reference to catalysis bychymotrypsin. In beginning my research program in1969, I resolved not to study chymotrypsin. It had beenchemically and kinetically analyzed in detail, and a crys-tal structure identified the catalytic triad of Ser195, His57,and Asp102. His57 functioned as a base, removing the�-OH proton of Ser195 as the �-oxygen attacked the sub-strate carbonyl group to form a metastable tetrahedralintermediate. Collapse of the intermediate with releaseof the N-terminal cleavage peptide produced peptidylchymotrypsin, which underwent hydrolysis to free chy-motrypsin and the C-terminal cleavage peptide. I re-garded chymotrypsin to be in good hands and did notsee how I could contribute.

The above mechanism explained everything, with oneexception. His57, displaying a pKa value of 7, seemed tooweak a base to abstract a proton from serine. Principlesof physical organic chemistry would mandate a base witha pKa value between those of the serine �-OH, pKa �13.6 (96), and the N terminus of the leaving cleavagepeptide, pKa � 9. The standard rationale was that thetransition state would embody the appropriate basicities,but there was no explanation for how this would bebrought about. A solution awaited experiments in 1972,1987, and the 1990s.

My colleague W. Wallace Cleland (Fig. 7), seeking toexplain low-deuterium fractionation factors ( 1.0) inseveral enzymatic mechanisms, published a paper in1992 suggesting the presence of LBHBs in certain en-zymes (97). Deuterium fractionation factors expressedthe equilibrium binding of deuterium relative to hydro-gen. Low factors indicated favorable hydrogen versusdeuterium binding. The cysteine �-SH displayed low-deuterium fractionation, but cysteine was absent in thesesystems. LBHBs also displayed low-deuterium fraction-

FIGURE 7. The author with W. Wallace Cleland (left) in 2007 in Wal-tham, Massachusetts.




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ation. Simultaneously, John A. Gerlt, now at the Univer-sity of Illinois, and Paul G. Gassman at the University ofMinnesota provided a theoretical basis for short-strongH-bonding in certain enzymatic mechanisms (98).

Cleland presented the deuterium fractionation evi-dence at a conference we attended, and on the flighthome, he commented on the unfavorable reception hereceived. I suggested that he find spectroscopic evidence.Cleland asked what kind of spectroscopy, and I suggestedNMR. In that moment, I recalled the work of Robillardand Shulman in 1972. They found a downfield 1H NMRsignal at 18 ppm in chymotrypsin at low pH values andassigned it to the proton bridging His57 and Asp102 (99).This proton resonated at 15 ppm at pH values above 7and at 18 ppm at low pH values. John L. Markley, then atPurdue University, independently extended this to tryp-sin (100). The 18 ppm signal for protonated His57

seemed consistent with an LBHB. I immediately realizedthat His57 would be protonated at and above pH 7 in themetastable tetrahedral intermediates for peptide hydrol-ysis. Could an LBHB stabilize these intermediates and re-lated transition states?

My Ph.D. preceptor, Professor Abeles, introduced pep-tidyl trifluoromethyl ketones as transition state analoginhibitors of serine proteases, including chymotrypsin.He and his associates found the methyl esters ofN-acetyl-phenylalanine, N-acetyl-glycyl-phenylalanine, N-acetyl-valyl-phenylalanine, and N-acetyl-leucyl-phenyl-alanine to be good substrates for chymotrypsin, with thebest being N-acetyl-leucyl-phenylalanine methyl ester.They synthesized analogous peptidyl trifluoromethyl ke-tones in which -CF3 replaced -OCH3, and they showedthat they formed tetrahedral adducts with Ser195

in chymotrypsin (101–103). The compounds were excel-lent inhibitors and analogs of the metastable tetrahedralintermediates, with the best being N-acetyl-leucyl-phe-nylalanyl-CF3, with a dissociation constant of 0.2 nM forthe ketonic (unhydrated) form. I thought that the ad-ducts would include protonated His57 and displayLBHBs. I called Abeles and asked if he had retained asample of the best inhibitor and explained my thoughts.He replied that he would find out and get back to me. Ithen found in the library that, in 1987, Liang and Abeles(103) had already published what I had in mind. In thecomplex of chymotrypsin with N-acetyl-leucyl-phenyla-lanyl-CF3, they found a downfield proton at 18.7 ppmthat persisted at pH 9. I called Abeles immediately andtold him that he had already done what I had planned,and he promptly recalled his and Liang’s earlier work. He

also sent me a sample of the inhibitor. We repeated thework and found the downfield signal at 18.9 ppm.

I and John Tobin proceeded to assign the downfieldprotons in protonated His57 of chymotrypsin and the tet-rahedral complex of chymotrypsin with N-acetyl-leucyl-phenylalanyl-CF3 as LBHBs, the only LBHBs assigned ina protein at the time (104). Simultaneously, W. WallaceCleland and Maurice M. Kreevoy at the University ofMinnesota elaborated on their theory of catalysis byLBHBs (105). With Tobin, we put forward in Fig. 8A amechanism for acylation of chymotrypsin and postulatedthat the LBHB stabilized the metastable tetrahedral in-termediate and, by Hammond’s postulate, the transitionstate (104). We noted the similarity of this tetrahedral in-termediate to the Ser195 adduct of N-acetyl-leucyl-phe-nylalanyl-CF3 (Fig. 8B) and emphasized that Liang andAbeles had found the downfield proton in this complexand shown it to persist above pH 9. We further pointedout that Richard Henderson at the Medical Research

FIGURE 8. LBHBs in chymotrypsin. A, role for the His57-Asp102 LBHB inthe acylation of Ser195 in chymotrypsin. B, LBHB in the tetrahedraladduct of chymotrypsin with N-acetyl-leucyl-phenylalanyl-CF3 (N-AcLF-CF3).




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Council had methylated His57 in chymotrypsin, decreas-ing activity by 2 � 105 (106). This chemical mutationdisrupted the LBHB, meaning that it could account for105 of the rate enhancement in chymotrypsin, corre-sponding to transition state stabilization of 7 kcal/mol.We further noted the downfield proton in cis-urocanicacid and suggested it as a “first approximation” to amodel for protonated His57 (104).

In Fig. 8A, we assigned partial charges y� to His57 andy� to Asp102, where 0.5 � y 1.0 (104). We did notknow the exact position of the proton in the LBHB anddefined y accordingly. A value of 0.5 for y would corre-spond to a symmetrical H-bond, which was unlikely. Avalue between 0.5 and 1.0 in an unsymmetrical LBHBseemed more likely. In the absence of a paramagneticmetal or a ring current effect, we thought the downfieldproton to be magnetically deshielded because of elonga-tion of the N�1–H bond due to the LBHB. Elongationcorresponded to pulling the proton away from the imid-azole ring, leaving increased electron density in the ringand increased basicity at the His57 N�3. In this way, thebasicity of His57 would increase in the approach to thetransition state, making it a suitable base to catalyze pro-ton transfer from Ser195.

The excellent review by Hibbert and Emsley (107) onstrong H-bonds in small molecules guided us in our as-signment. They defined three classes of H-bonds: con-ventional weak H-bonds (10 kcal/mol), intermediatestrong H-bonds (10 –20 kcal/mol), and very strongH-bonds (�24 kcal/mol). The authors pointed out thatthe intermediate strong H-bonds could form betweenheteroatoms with similar proton affinities, and they dis-played downfield proton chemical shifts (�H 18 –20ppm), low-deuterium fractionation factors, deuteriumisotope effects on NMR chemical shifts, and linearH-bonding with less than van der Waals contact dis-tances separating heteroatoms. We adopted these prop-erties as distinguishing features of LBHBs.

With associates Constance S. Cassidy and Jing Lin andWisconsin colleague John L. Markley, we found corrobo-rative evidence for the LBHB in peptidyl trifluoromethylketone complexes of chymotrypsin (108 –112). Thedownfield proton chemical shifts were 18.6 –18.9 ppmdepending on peptidyl structure. The deuterium frac-tionation factors were 0.3– 0.4. Activation enthalpies forchemical exchange of the LBHBs with water were 14.7–19.4 kcal/mol, �10 kcal/mol higher than for conven-tional H-bonds. The LBHBs displayed deuterium andtritium isotope effects on chemical shift. These were the

expected properties for the intermediate strong H-bondsdefined by Hibbert and Emsley (107).

Catalytic Significance of the LBHB—As evidence of thesignificance of LBHBs, the chymotrypsin adducts withN-acetyl-phenylalanyl-CF3, N-acetyl-glycyl-phenylala-nyl-CF3, N-acetyl-valyl-phenylalanyl-CF3, and N-acetyl-leucyl-phenylalanyl-CF3 displayed pKa values for His57

from 10.7 for N-acetyl-phenylalanyl-CF3 to 12.1 forN-acetyl-leucyl-phenylalanyl-CF3. In linear free energycorrelations, plots of literature values of log(kcat/Km) forpeptidyl methyl esters against pKa or �H for the corre-sponding peptidyl trifluoromethyl ketones proved to belinear with positive slopes. Plots of log Ki for peptidyl tri-fluoromethyl ketones against �H or pKa for the same in-hibitors proved to be linear with negative slopes.

The difference in pKa for His57 in the tetrahedral com-plex of chymotrypsin�N-acetyl-leucyl-phenylalanyl-CF3

(pKa � 12) and free chymotrypsin (pKa � 7) corre-sponds to a free energy difference of 7 kcal/mol, thesame as the difference in activation energies for free chy-motrypsin and N-methylated His57 chymotrypsin. Thus,the physicochemical properties of peptidyl trifluoro-methyl ketone adducts support the proposition of theimportance of the LBHB in catalysis. All evidence pointsto the role of the LBHB in elevating the pKa of His57 tobetween 9 and 13 at the tetrahedral intermediate, mak-ing it the ideal base catalyst for serine protease action.

Analogous evidence for complexes of peptidyl trifluo-romethyl ketones with the serine protease subtilisin in-cluded downfield �H and low-deuterium fractionationfor the His-Asp contact (113). Further evidence showedthe contact distance between the His57 N�1 and Asp102

O�1 of the complex of chymotrypsin with N-acetyl-leu-cyl-phenylalanyl-CF3 to be less than the van der Waalscontact distance (114). In addition, the crystal structureof subtilisin at atomic resolution clearly showed a length-ening of the His57 N�1H–H bond (115).

LBHBs in Small Molecules—Constance S. Cassidy, JohnTobin, Jing Lin, and Mary Cloninger in our group under-took studies of LBHBs in small molecules. Physical andphysical organic chemists had defined the physicochemi-cal properties of LBHBs in small molecules (107).

Fig. 9 illustrates energy profiles for weak, intermediatestrength, and very strong H-bonds. Weak H-bonds, as inwater, display double-minimum energy profiles sepa-rated by high barriers. Intermediate strength H-bonds,or LBHBs, are shorter than allowed by van der Waalsradii, and the barrier is lowered to near the zero-point vi-brational energy, as in the complex of N-methylimida-zole (N-MeIm) with 2,2-dichloropropionate. This com-




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plex (�H � 18 ppm) displays an anti-symmetrical C–Ovibrational frequency lying between those of analogousion pairs (N-MeImH�–�OOC-R) formed with strongeracids and those of neutral complexes (N-MeIm–HOOC-R formed with weaker acids) (116). Both ionpaired and neutral complexes display chemical shifts up-field from 18 ppm. The spectroscopic properties of the18 ppm complex are as expected for an LBHB.

Associate Mary Cloninger showed that the down-field intramolecular H-bond in cis-urocanic acid canbe altered by introducing steric bulk at C2. The His57-Asp102 contact in chymotrypsin is sterically com-pressed in x-ray crystal structures of peptidyl trifluo-romethyl ketone complexes (114). By the foregoinghypothesis, the basicity of His57 (pKa � 12) can be at-tributed to this contact. The imidazole basicities of2-substituted cis- and trans-urocanates in water sup-port this hypothesis, as shown in Structure 1 (117).The increasing sizes of the 2-chloro and 2-bromo sub-stituents increase the pKa in cis-urocanates but not inthe trans-isomers. The electronic effects of chlorineand bromine cannot account for the results, so thesteric effects must be invoked. The increased basicitycan be ascribed to the increased strength of theH-bond and alternatively by increased electrostatic in-teraction in the contact. In either case, the closer con-tacts in the cis-2-chloro- and 2-bromourocanates leadto increased base strength in the imidazole ring. Thislikely occurs in the chymotrypsin transition states.

Strong H-bonds in Aqueous/Organic Media—Our groupaddressed widespread reservations as to whether LBHBscould be present in aqueous media (118). We chose tostudy intramolecular LBHBs in sterically enforced

H-bonding within molecules, such as hydrogen maleatemonoanion (H-maleate) in Fig. 9B, perhaps the mostthoroughly studied strong H-bond in organic chemistry.Typical research of this molecule involved crystal struc-tures or strictly anhydrous, aprotic solvents in solution.Under these conditions, H-maleate served as an exampleof a single-well H-bond. Crystal structures showed theH-bond to be nearly symmetrical, and perfectly symmet-rical in the imidazolium salt (119). The downfield protonin anhydrous solvents appeared at �20 ppm.

Theoretical calculations put the energy of the H-bondvariously at �24 kcal/mol in vacuum. McAllister (120)studied the effect of medium polarity and found theH-bond energy to be �20 kcal/mol in “a dielectric con-tinuum” similar to water. In aqueous solution, the differ-ence in pKa values of 1.9 and 6.2 for maleic acid couldnot be accounted for on purely electrostatic grounds, butan energy of 12–15 kcal/mol for the intramolecularH-bond could explain the difference (118).

Why then would the LBHB in H-maleate not appear inaqueous solutions? We reasoned that it might undergoexchange too quickly with protons in water to appear inthe NMR spectrum. Therefore, Constance S. Cassidy,

FIGURE 9. Three classes of hydrogen bonds: weak, low-barrier, and single-well.

STRUCTURE 1




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John Tobin, and Jing Lin in our group studied the NMRspectra of H-maleate in 90:10 (v/v) acetone-d6/H2O at�50 °C to decrease the exchange rate (121). These solu-tions contained 0.4 mol fractions of water molecules(40%). We also studied other sterically strained dicar-boxylate monoanions, H-2,2-dimethyl malonate, andH-cis-cyclohexane 1,2-dicarboxylate in the same solvent,and we found the downfield signals at 20.2, 19.0, and19.2 ppm, respectively. Therefore, intermediate strongintramolecular H-bonds exist in organic/aqueous media.Substrate-binding sites in enzymes clearly are not aque-ous, although water has access at the surface. The ace-tone-d6/H2O likely allows more access to water than abinding site in a protein. In further work on the chemicalproperties of intramolecular H-bonding, Lin in ourgroup showed the activation energy for exchange withmedium protons and deuterium fractionation to be com-patible with an LBHB (122)

Nearly all studies of ultra strong H-bonds in chem-istry employed non-biological molecules. A recentstudy showed that all of the acid-base groups in proteinscan form ultra strong H-bonds (123).

At the time we made our assignments of LBHBs (104),I told my associates that the worst reaction we could ex-pect would be for other investigators simply to acceptour claim. This would signal the end of research onLBHBs in serine proteases. I need not have been con-cerned. Many objections followed, including the theorythat strong H-bonds could not exist in water. This madeit necessary to carry out much more detailed and criticaltests. The results outlined above fully corroborated ouroriginal claim.

Acknowledgments—I am exceedingly grateful to my graduate and post-doctoral students for sharing the thrill of discovery with me. I am alsograteful to senior investigators in Asia, Europe, and the Americas forvaluable collaborations. I am equally grateful to the National Institute ofDiabetes and Digestive and Kidney Diseases and the National Institute ofGeneral Medical Sciences of the National Institutes of Health for contin-uously supporting my research through predoctoral fellowships from1964 to 1967, a postdoctoral fellowship in 1968, and individual researchgrants from 1969 to my retirement in 2008.

Address correspondence to: [email protected].

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Perry Allen Frey2008−Transient Intermediates in Enzymology, 1964

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http://www.jbc.org/lookup/doi/10.1074/jbc.X115.650879

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;290/17/10610&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/290/17/10610

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=290/17/10610&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/290/17/10610

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/290/17/10610.full.html#ref-list-1

http://www.jbc.org/

transient intermediates in enzymology, 1964 –2008

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