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Proc. Natl. Acad. Sci. USA Vol. 82, pp. 2225-2229, April 1985

Alternative view of enzyme reactions ( effects/nucleophilic substitution) MICHAEL J. S. DEWAR AND DONN M. STORCH* Department of Chemistry, The University of Texas, Austin, TX 78712 Contributed by Michael J. S. Dewar, November 13, 1984

ABSTRACT Since adsorption of the in the active reaction is an example, the corresponding minimum site of an enzyme can occur only if all solvent is squeezed out reaction path (MERP) in the gas phase being (4-7) of the from between them, any reaction between them takes place in type indicated in Fig. 1, path a. In the gas phase, where the the absence of any intervening solvent-i.e., as it would in the energy liberated in forming the initial adduct cannot be rap- gas phase. Recent work has shown that ionic reactions in the idly dissipated, the reaction may take place without activa- gas phase often differ greatly from analogous processes in so- tion by a "hot " mechanism, as indicated by the lution. Therefore, current interpretations of enzyme reactions dotted line in Fig. 1, path a. In , the initial gain in in terms of solution chemistry are misguided. The large rates energy is eliminated (7, 8) leaving a conventional and specificity of enzyme reactions may be due simply to elimi- barrier (Fig. 1, path b). nation of the solvent. The cleavage of by chymotryp- Gas-phase reactions of the second type follow (7, 9, 10) sin and A can be interpreted satisfactorily in the course indicated by the schematic MERP labeled c in this way. Fig. 1. The reactants combine exothermically and without activation to form a stable adduct. Any activation barrier to The basic problem in enzyme chemistry is to explain why such a solvactivated reaction in solution must then be due enzyme reactions are so fast and so specific. Enzymes pro- entirely to hindrance by the solvent, in particular to the fact mote reactions of their substrates more efficiently by many that solvent must be removed from the before the other orders of magnitude than other catalysts, while reactions of reactant can approach. The then corresponds other analogous are catalyzed far less efficiently, to a solvated ion-dipole complex, little or no change in bond- if at all. (We will use the term "substrate" only in the limited ing having taken place. Since the adduct is larger than the sense indicated above.) reactant ion, its solvation energy should be correspondingly Since there are no analogs of enzymes, attempts to explain smaller, so it is likely to become a high-energy intermediate, their activity have been of an ad hoc nature, postulating a the MERP being as indicated by path d in Fig. 1. cooperation of different effects for which there is no evi- Calculations and experiment (see ref. 9) suggest that sub- dence (1) or assuming that adsorption of the substrate can stitution at carbonyl carbon by anions, in particular hydroly- to unprecedented changes in the of activation sis and alcoholysis of esters and amides by or al- (2). Attempts to explain the specificity of enzyme reactions koxide , follow this pattern. Other reactions that seem to are mostly based on Koshland's induced-fit theory (3), for be solvactivated are the SN2' reaction (7) and nucleophilic which again there are no analogies. substitution on silicon (10). The of an enzyme is a cleft or hollow into which Since adsorption of the substrate in the active site of an its substrate fits closely. Adsorption of the substrate can enzyme to total exclusion of solvent () from be- then take place only if all molecules of solvent (i.e., water) tween them, any subsequent reaction between them takes are squeezed out from between them. Thus, the enzyme re- place in the absence of solvent-i.e., as it would in the gas action takes place in the absence of solvent, just like an anal- phase. The only difference is that any energy liberated in the ogous reaction in the gas phase. Since gas-phase reactions initial association can be rapidly dissipated, so "hot mole- often differ drastically from analogous reactions in solution, cule" processes are no longer possible. Autoactivated reac- discussions of enzyme reactions clearly should be based on tions consequently encounter activation barriers analogous analogies with the former. Yet all past and current mechanis- to those in solution. However, a solvactivated reaction will tic studies of enzyme reactions have assumed them to be take place without activation. Since the activation analogs of reactions in solution. This cannot but have led to for the hydrolyses of amides by water, in the absence of ac- misconceptions and errors. ids or bases, are usually >>100 kJ/mol, removal of the acti- Our purpose here is to show that the "problems" indicated vation barrier would be expected to increase the rate by at above become nonproblems if examined from this alterna- least 20 orders of magnitude. This is more than enough to tive viewpoint. account for the whole of the acceleration observed in the of appropriate peptides by peptidases such as Activation Barriers in Solution: Autoactivated and . "Solvactivated" Reactions There are, however, logistic problems to be overcome in implementing this scheme. How is the necessary anionic Recent work (4-10) indicates that reactions of ions with neu- center generated in the active site of the enzyme, given that tral molecules can be divided into two groups. In an autoac- reactions ofthis kind normally take place in neutral solution? tivated reaction, the reactants combine exothermically in the And second, if such an anionic center were present, would gas phase to form a charge-dipole adduct, separated by a not its desolvation require as much energy as desolvation of conventional activation barrier from the products. The SN2 the anion in a corresponding reaction in solution?

The publication costs of this article were defrayed in part by page charge Abbreviation: MERP, minimum energy reaction path. payment. This article must therefore be hereby marked "advertisement" *Present address: U.S. Air Force Academy, Colorado Springs, CO in accordance with 18 U.S.C. §1734 solely to indicate this fact. 80840.

2225 Downloaded by guest on September 28, 2021 2226 Biochemistry: Dewar and Storch Proc. NatL Acad Sci. USA 82 (1985) CH3CONH2 + H20 -O CH3COOH + NH3 AH, 8.4 kJ/mole CH3CONHBu + H20 -* HCOOH + C4H9NH2 AH, 22.4 kJ/mole HCONMe2 + H20 -O HCOOH + HNMe2 (b)Y RX AH, 36.0 UJ/mole. Water has little effect on the overall heat of reaction, the cW YRYRX (b) hydrolyses of peptides in aqueous solution being close to 0~~~~~ thermoneutral (17). Any catalytic process must be revers- ible. Under suitable conditions chymotrypsin can indeed cat- X-+M alyze the formation of amides from amines and (17). I Peptides are hydrolyzed under biological conditions because of the gain in entropy, one molecule of reactant () being converted into two of products. Since the adsorbed substrate is held firmly in place in the (d) active site with the reacting groups appropriately oriented, the reaction is effectively unimolecular, so the correspond- ing Arrhenius preexponential factor should be =1014. Since Reaction Coordinate the corresponding first-order rate constants seem to be ca. 102 (11, 12), the overall is ca. 60 kJ/mol. FIG. 1. Schematic minimum energy reaction paths for ion-mole- Our object is to show that, in the absence of solvent, the cule reactions: paths a and b, an SN2 reaction; paths c, d, and b, a four reactions (A-D) can take place fast enough to account solvactivated reaction; paths a and c, reactions in the gas phase; for the rates of chymotrypsin-catalyzed cleavage of pep- paths b and d, reactions in solution. The dotted line in path a indi- tides, without any need to postulate additional factors. The cates the course of a "hot molecule" reaction. effect of the enzyme could then be attributed wholly to ex- clusion of solvent. Since the hydrolysis takes place in four The Mechanism of by Chymotrypsin steps and is almost thermoneutral, the activation energy of each step must be _60 kJ/mol, and none must be strongly The structure of chymotrypsin and of its active site and the endothermic. reactions involved in the catalytic hydrolysis of peptides by No thermochemical data are available for tetrahedral ad- it seem to have been established (11, 12). The hydrolysis ducts from carboxyl derivatives. We are forced to estimate takes place in four steps, each involving the addition of an them theoretically. Since no current procedures can be ap- anion (X-) to the carbonyl group of a carboxylic in plied to enzymes, the calculations are confined to models the substrate (RCOY) or the reverse of such a process: simulating their active sites. Use of any adequate ab initio R procedure, even in this connection, would moreover be X- + RCOY ;± X-C-0- [1] wholly impracticable. The only practicable models of ade- Y quate performance are MINDO/3 (18), MNDO (19), and The relevant anions are generated by proton transfer from AM1 (20), which give (21, 22) results comparable with those HX to the carboxylate group of Asp-102 via the from good ab initio ones at less than 1/1000th the cost in ring of His-57 ("charge relay mechanism"): computing time. Here AM1 was the obvious choice because it alone reproduces bonds in a reasonably satisfac- (Asp)COO- H-N N H-X +± (Asp)COOH N NH X-. [2] tory manner (20). A proper model of chymotrypsin would include not only [Recent criticisms (13, 14) of this mechanism are refuted be- the groups directly involved in the reaction but also adjacent low.] The groups involved in each of the four steps (A-D) are polar groups that can interact with them, either by hydrogen as follows, the arrow indicating the direction in which Eqs. 1 bonding or electrostatically. Calculations based on such a and 2 operate: model would represent a major undertaking, even using AMi. Indeed, even the systems indicated in Eq. 1 would tax A(--) X = (Ser-195); Y = NHR' our current computer facilities. In this preliminary investiga- tion, we studied only simple models of the unit processes B(+-) X = NHR'; Y = (Ser-195) involved. More detailed calculations are planned, and a de- will be C(-*) X = HO; Y = (Ser-195)O tailed account of those summarized below presented elsewhere. D(+-) X = (Ser-195); Y = HO. Since the intervening imidazole ring remains virtually un- changed during the charge relay, it can be ignored. Formic RCOY is the peptide (RCONHR') in step A, the Ser-195 es- seemed a suitable model for since their ter of RCOOH in steps B and C, and the acid (RCOOH) itself pKas (23) are similar (3.75 vs. 3.86). or was in step D. HX is Ser-195 in A and D, the amine (R'NH2) used as a model for ; N-methylformamide or N-methyl- derived from the peptide in C, and water in D. The reaction acetamide, for the substrate; methylamine, for the amine of Eq. 1 takes place in steps A and D from left to right and in formed by its cleavage; and methyl formate or methyl ace- steps B and D from right to left. tate, for the intermediate serine ester. The results quoted Thermochemical data (15, 16) indicate that hydrolyses of here were for the combination methanol/N-methylforma- simple amides in the gas phase are weakly endothermic- mide/methylamine/methyl formate. The results for other e.g., choices differed only by a few kilojoules per mole. All geom- etries were fully optimized. Calculations were carried out by HCONH2 + H20 -* HCOOH + CH3NH2 using the MOPAC program, available from the Quantum AH, 2.9 kJ/mole Chemistry Program Exchange. Downloaded by guest on September 28, 2021 Biochemistry: Dewar and Storch Proc. NatL. Acad. Sci. USA 82 (1985) 2227

Since the charge relay mechanism involves transfer of a ofthe enzyme-catalyzed reaction is due entirely to the exclu- proton from an to a carboxylate ion, it is naturally sion of solvent. Indeed, the efficiency with which chymo- very endothermic. The calculated [observed (15, 16)] heat of catalyzes the hydrolysis of suitable peptides can be reaction for our model system is 128.4 (142.3) kJ/mol. The attributed to an ingenious expedient that enables the enzyme active site of chymotrypsin is therefore initially neutral. The to carry out a gas-phase reaction in aqueous solution. substrate can be adsorbed, and water eliminated, without hindrance because the active site of chymotrypsin carries no Role of Solvactivated Reactions in Enzyme Chemistry ionic charges. The anionic nucleophile is generated only af- ter the substrate has been adsorbed, in response to the de- Similar principles seem likely to hold generally in enzyme mand of the latter for anions. Desorption of the products chemistry, the extravagant rates of enzyme reactions being likewise takes place without hindrance because the negative due simply to the fact that they take place in the absence of charge is withdrawn to the aspartate group in chymotrypsin, solvent, under conditions equivalent to the gas phase. If so, leaving the active site neutral, before desorption takes place. the basic steps in enzyme reactions must be solvactivated This explains how the enzyme overcomes the difficulty not- because the activation barrier of an autoactivated process ed earlier. The anionic center in the active site is developed survives even in the absence of a solvent. The virtual ab- only after the substrate has been adsorbed. sence of pericyclic reactions and of the SN2 reaction from The heats of reaction (kJ/mol) calculated (AM1) for our lists of enzymatic processes is particularly significant in this models of the individual steps were as follows: A, 50.2; B, connection. Almost the only exception is , a -37.7; C, -31.4; D, 7.5. process essential in and one that cannot easily be The values shown above are of course only approximate, brought about other than by the SN2 route. This reasoning having been derived by an approximate method from a high- leads to three hypotheses that may serve as general guides. ly simplified model. The differences between the calculated HYPOTHESIS I. Enzyme reactions take place at the same heats of reaction are nevertheless large enough to make it rate as analogous gas-phase reactions, apartfrom the possi- likely that the first step (A) is the most endothermic and, bility of "hot molecule" processes in the latter. therefore, rate determining. The experimental evidence sug- HYPOTHESIS II. Reactions used by enzymes are usually gests that this is indeed the case (24). It is the most endother- ones that involve no intermediate barriers in the gas phase. mic because nucleophilic addition to the carbonyl group in HYPOTHESIS III. Any charged group actively involved in an amide is harder than analogous addition to a carboxylic an enzyme reaction must be generated by some kind ofrelay acid or ester due to the greater resonance stabilization of mechanism after the substrate has been adsorbed. Charged amides. Esters related to poor chymotrypsin substrates are groups can be present initially in an active site only if they often hydrolyzed by chymotrypsin as fast as its true sub- correspond to charged groups of opposite sign in the sub- strates (11, 12). Presumably the lesser endothermicity of the strate, the coulombic attraction between them being needed first step in the case of an ester compensates for retardation to squeeze out solvent. by odd molecules of water remaining in the active site. Some applications of these ideas are indicated below. In step A, the negative charge in the reactant resides on Reactivation of . Cholinesterase catalyzes Asp-102, approximately half a unit on each , one oxy- (11) the hydrolysis of by a charge-relay mecha- gen being hydrogen-bonded to His-57 and the other to the nism similar to that used by chymotrypsin. Nerve gases inac- hydroxyl of Ser-214 (11). In the , the charge is con- tivate cholinesterase by phosphorylating the relevant serine centrated on the oxygen of the amide group undergoing hy- moiety, the resulting esters not being easily hydrolyzed. The drolysis, which is hydrogen-bonded to the imino groups of problem is to explain how such relatively inert phosphorus Ser-195 and gly-193 (11). Therefore, hydrogen bonding compounds can react with cholinesterase at an almost diffu- should not greatly alter the heat of reaction since the hydro- sion-controlled rate and yet form esters that are again rela- gen bonds in the reactants and products are formed by analo- tively inert. Furthermore, the only nucleophiles that do react gous . The same argument also applies to the subse- rapidly with the serine esters, thus regenerating the enzyme, quent steps. are oximes, compounds which do not usually show unusual Since the strength of a hydrogen bond is roughly propor- reactivity as nucleophiles. tional to the negative charge on the donor atom, the overall The reactions of nerve gases with anions must clearly be stabilization by hydrogen bonding should change linearly solvactivated. This is why they react so rapidly with cholin- with transfer of charge as the reaction proceeds. This repre- , provided of course that their molecules are of the sents another basic difference from analogous ionic reac- right shape. Since they are only weakly polar, the solvactiva- tions in solution. Solvation of an ion in solution involves ori- tion barriers to their reactions with anions in solution will be entation of surrounding molecules of solvent by electrostatic large (11, 12). This is why their reactions with nucleophiles interactions with the ion. Dispersal of charge, in forming a in solution are so slow. However, once a nerve gas has react- transition state, leads to a decrease in such orientation and, ed with cholinesterase to form the corresponding serine es- hence, to a decrease in solvation energy. This is not the case ter, problems arise in hydrolyzing it. The hydrolysis, being in an enzyme reaction because the interacting groups in an of SN2 type, requires the nucleophile to attack trans to the enzyme-substrate adduct are fixed both in number and in serine oxygen, as opposed to lateral attack involved in the orientation. case of a carboxylic ester. Water, hydrogen-bonded to the The activation energy of each individual step (A-D) will , is unable to reach. To cleave the ester, one needs a naturally be greater than its endothermicity. Moreover, each water substitute-a molecule containing a crescent-shaped is a multibond process, and multibond reactions are "forbid- unit with an acidic hydrogen at one end to hydrogen bond to den" (25)-i.e., they do not normally take place in a syn- the histidine and a nucleophilic group at the other end to chronous manner. However, migrations of hydrogen repre- attack phosphorus. Oximes meet these conditions perfectly. sent an exception to the rule (25) because hydrogen is adept Note that according to this interpretation, it is the nitrogen of at forming three-center bonds and also may undergo rapid the oxime that attacks phosphorus. migration by tunneling. Hydrogen migration can occur, . Senine peptidases cleave peptides by therefore, in concert with other processes without much base-catalyzed hydrolysis, using the same change in the overall activation energy. (Glu-His-Ser) as chymotrypsin. Carboxypeptidase A (2, 11, The results reported here are consistent with our suggest- 12) seems to act by acid-catalyzed hydrolysis, this being ed mechanism. It seems reasonable to suppose that the rate needed because it specifically cleaves the carboxyl-bearing Downloaded by guest on September 28, 2021 2228 Biochemistry: Dewar and Storch Proc. NatL Acad Sci. USA 82 (1985) terminal groups from peptides. The terminal carboxyl is ion- solution (27). Thus, while toluene in solution is a weaker acid ized under biological conditions, and the resulting negative than water by ±25 pKa units, in the gas phase it is stronger. charge would inhibit nucleophilic attack on the adjacent am- Therefore, carbanions stabilized by only a little ide link. more effective than phenyl could be formed as intermediates The active site of carboxypeptidase A contains a gluta- in enzyme reactions. mate ion (Glu-270) and a ion, the latter being attached to One of the major carbon-carbon bond-forming reactions another glutamate residue (Glu-72), the imidazole rings of in biological systems is the Claisen condensation. However, two histidine units (His-69 and His-196), and a molecule of it has been difficult to account for the formation of the nec- water. Currently accepted mechanisms (2, 11, 12) for car- essary carbanions formed under enzymatic conditions. In- boxypeptidase A assume the zinc to act as a Lewis acid, deed, Arnstadt (28) has claimed that free carbanions are not coordinating to the amide carbonyl and, thus, promoting nu- involved in enzymatic processes of this kind because no ex- cleophilic addition to it. The adduct formed by carboxypepti- change with 2H20 took place during them. Both of these dase A with glycyl-L-, a poor substrate, has such a problems disappear in the light of the present discussion. structure (26). The attacking nucleophile originally was as- Since water is excluded from the active site during a "true" sumed to be Glu-270, leading to a mixed anhydride-i.e., enzyme reaction, carbanions should be formed with corre- sponding ease, while the absence of water, of course, would R, R, make exchange impossible. Carbanions also may play an unsuspected role in oxida- (Glu-270)COO- C=O-Zn+-- (Glu-270)COO-C-O-ZnH tion-reduction processes by acting as donors, quite apart from their possible role as nucleophiles or equivalents R2NH R2NH of organometallic species. Reductions by hydride transfer (Glu-270)COOCOR, + R2NH2 + Zn+. [3] are well recognized in reactions mediated by flavin or nico- tinamide coenzymes. Carbanions could be even more effec- This mechanism would, however, violate Hypothesis III. tive. For example, they may play a role in the reduction of Indeed, Breslow et al. (26) have shown that the anhydride is dinitrogen (N2) by . Current mechanisms assume not an intermediate. They suggest that the nucleophile is, in this to take place by a series of steps, each involving reduc- fact, a molecule of water hydrogen-bonded to Glu-270, the tion by electron transfer, followed by . The pos- acid being formed directly rather than by hydrolysis of an sibility of reduction by hydride transfer, rather than electron anhydride. transfer, does not seem to have been considered. In the reverse reaction, where the enzyme catalyzes amide Potentials. The differences between relative acid- formation from an acid and an amine, this mechanism would ities in solution and in the gas phase are due primarily to the involve displacement of the water bonded to zinc by the car- different energies of solvation of the (ionic) conjugate bases. bonyl group of the acid. Since carboxylic acids are much Similar differences should occur in any reaction where a weaker bases than water and since the zinc carries a positive neutral molecule is converted to an anion, in particular elec- charge, the displacement would be endothermic and proba- tron-transfer processes. Therefore, differences between re- bly also would encounter a considerable solvactivation barri- dox potentials in solution and in the gas phase should parallel er. It seems more likely that the catalyst is not the zinc itself, those in the pKa of acids. Reductions involving the forma- acting as a Lewis acid, but the molecule of water attached to tion of carbanions should take place, for example, much zinc, acting as a protic acid: more easily in the gas phase (or during an enzyme reaction) than in solution. Therefore, attempts to assess electron- O@H transfer mechanisms for enzyme reactions from convention- R1 al redox potentials may prove to be misleading. (Glu-270)C- 0 >C=O..HO-Zn+ "Artificial Enzymes." The conclusions reached here are of O-/ R2NH H obvious relevance to attempts to synthesize "artificial en- 0ATH zymes." If such a species is to bring about rate enhance- ments comparable with those due to enzymes, it is not suffi- -- (Glu-270)COOH HO-C-OH HOZn. cient for it to contain the necessary reactive groups in the right orientation. The "active site" in the R2NH also must fit the substrate closely enough to ensure that ad- sorption of the latter can occur only if all solvent is extruded If so, peptides that displace the water from zinc will not be from between them. If an ion is involved in the reaction, it hydrolyzed, at least not by the "proper" route. This would must also be formed only after the substrate has been ad- explain why glycyl-L- is such a poor substrate. When it sorbed. Attempts to promote reactions in this way are, is adsorbed by carboxypeptidase A, the vital molecule of wa- moreover, likely to prove abortive unless they are of the solv- ter is lost. activated type. Nobody seems as yet to have suggested this mechanism as a possibility, probably because zinc in water behaves as a Implications Concerning Methodology base or a very weak acid. This, however, raises another ma- jor difference between gas phase and solution chemistry. The arguments above have general implications concerning The relative strengths of acids and bases in the gas phase are the methodology to be followed in studying enzyme reac- often quite different from those in solution (27), and inorgan- tions. ic represent one of the more striking examples. (i) Studies of enzymes with their active sites full of water Thus, silicic acid and aluminum hydroxide, both very weak have no relevance to their reactions with substrates. In par- acids in water, act as superacids in the gas phase, where they ticular, attempts (cf. ref. 13) to assess the possibility of pro- consequently serve as useful acid catalysts. It is, therefore, ton transfers from pKa measurements under such conditions entirely possible that the molecule of water attached to Zn' are meaningless because the relative strengths of acids are in carboxypeptidase A may be strongly acidic, so long as altered radically by removal of solvent. additional water is not present. (ii) Similar remarks apply to attempts to determine the Possible Role of Carbanions in Enzyme Reactions. Hydro- mechanisms of enzyme reactions from studies of analogous carbons are also much stronger acids in the gas phase than in reactions of "poor substrates." The latter react more slowly Downloaded by guest on September 28, 2021 Biochemistry: Dewar and Storch Proc. Natl. Acad. Sci. USA 82 (1985) 2229

simply because they do not fill the active site of the enzyme. 6. Wolfe, S., Mitchell, D. J. & Schlegel, H. B. (1981) J. Am. Such reactions are indeed analogs of reactions in solution Chem. Soc. 103, 7692-76%. and so bear no necessary relation to the true enzymatic pro- 7. Dewar, M. J. S. & Carrion, F. (1984) J. Am. Chem. Soc. 106, cesses. 3531-3539. 8. Chandrasekhar, J., Smith, S. F. & Jorgensen, W. L. (1984) J. (iii) The use of enzyme-inhibitor adducts as models of the Am. Chem. Soc. 106, 3049-3050. enzyme-substrate combination is also unsatisfactory for 9. Dewar, M. J. S. & Storch, D. M. (1984) J. Chem. Soc. Chem. similar reasons. Unless the inhibitor fills the active site Commun., in press. closely, it will not be a good model for a real substrate. There 10. Dewar, M. J. S. & Healy, E. (1982) Organometallics 1, 1705- is also the danger that the inhibitor may be an inhibitor sim- 1708. ply because it reacts with the active site in a manner different 11. Blackburn, S. (1976) Enzyme Structure and Function (Dekker, from the substrate. New York). (iv) Any approach to enzyme reactions along the lines 12. Ferscht, A. R. (1977) Enzyme Structure and Mechanism (Free- suggested here depends on the availability of information man, San Francisco). in the ab- 13. Bachovchin, W. W. & Roberts, J. D. (1978) J. Am. Chem. concerning the energetics of reactions complete Soc. 100, 8041-8047. sence of solvent. Experimental data of this kind are scanty, 14. Kossiakoff, A. A. & Spencer, S. A. (1981) Biochemistry 20, partly because techniques have been developed only recent- 6462-6474. ly for studying ion-molecule reactions in the gas phase and 15. Cox, J. D. & Pilcher, G. (1970) Thermochemistry of Organic partly because the demand for such information has been and Organometallic Compounds (Academic, New York). limited. Certainly no one has suggested that it could be of 16. Pedley, J. B. & Rylance, J. (1977) Sussex-N.P.L. Computer major interest to . Analysed Thermochemical Data: Organic and Organometallic (v) The arguments presented here suggest that theoretical Compounds (Sussex Univ., Brighton). calculations could* play a very effective role in enzyme 17. Fruton, J. S. (1982) Adv. Enzymol. 53, 239-307. refer to re- 18. Binkham, R. C., Dewar, M. J. S. & Lo, D. H. (1975) J. Am. chemistry, given that such calculations normally Chem. Soc. 97, 1285-3111. actions of isolated molecules. Calculations for effective 19. Dewar, M. J. S. & Thiel, W. (1977) J. Am. Chem. Soc. 99, models of enzymes have seemed far out of reach in the past 4899-4917. because of the lack of a suitable theoretical procedure. As 20. Dewar, M. J. S., Healy, E. F., Stewart, J. J. P. & Zoebisch, indicated above, AM1 seems to meet this need. We hope it E. G..(1985) 1; Am. Chem. Soc., in press. will prove as useful in enzyme chemistry as MINDO/3 and 21. Dewar, M. J. S. & Ford, G. P. (1q79) J. Am. Chem. Soc. 101, MNDO have already done in studies of organic reactions. 5558-5561. 22. pewar, M. J. S. & Storch; D. (1985) J. Am. Chem. Soc., in press. This work was supported by the Air Force Office of Scientific 23. Weast, R. C., Astle, M. J. & Beyer, W. H., eds. (1984) CRC Research (Contract F49620-83-C-0024) and the Robert A. Weleh Handbook of Chemistry.and Physics (CRC Press, West Palm Foundation (Grant F-126). Beach, FL), 54th Ed., D-166. 24. Dewar, M. J. S. (1984) J. Am. Chem. Soc. 106, 209-219. 1. Lipscomb, W. N. (1982) Acc. Chem. Res. 15, 232-238. 25. Hunkapiller, M. W., Forgac, M. D. & Richards, J. H. (1976) 2. Jencks, W. P. (1982) J. Biol. Chem. 257, 10893-10907. Biochemistry 15, 5581. 3. Koshland, D. E., Jr. (1%3) Cold Spring Harbor Symp. Quant. 26. Breslow, R. & Wernick, D. (1976) J. Am. Chem. Soc. 98, 259- Biol. 28, 473-482. 261. 4. Olmstead, W. N. & Brauman, J. I. (1977) J. Am. Chem. Soc. 27. Bowers, M. T., ed. (1979) Gas Phase Ion Chemistry (Academ- 99, 4219-4228. ic, New York), Vol. 2. 5. Keil, F. & Ahlrichs, R. (1976) J. Am. Chem. Soc. 98, 4787- 28. Arnstadt, K.-I., Schindbleck, G. & Lynen, F. (1975) Eur. J. 4793. Biochem. 55, 561. Downloaded by guest on September 28, 2021