Proc. Natl. Acad. Sci. USAVol. 82, pp. 2225-2229, April 1985Biochemistry

Alternative view of enzyme reactions(solvent 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 substrate in the activesite of an enzyme can occur only if all solvent is squeezed outfrom between them, any reaction between them takes place inthe absence of any intervening solvent-i.e., as it would in thegas phase. Recent work has shown that ionic reactions in thegas phase often differ greatly from analogous processes in so-lution. Therefore, current interpretations of enzyme reactionsin terms of solution chemistry are misguided. The large ratesand specificity of enzyme reactions may be due simply to elimi-nation of the solvent. The cleavage of peptides by chymotryp-sin and carboxypeptidase A can be interpreted satisfactorily inthis way.

The basic problem in enzyme chemistry is to explain whyenzyme reactions are so fast and so specific. Enzymes pro-mote reactions of their substrates more efficiently by manyorders of magnitude than other catalysts, while reactions ofother analogous molecules are catalyzed far less efficiently,if at all. (We will use the term "substrate" only in the limitedsense indicated above.)

Since there are no analogs of enzymes, attempts to explaintheir activity have been of an ad hoc nature, postulating acooperation of different effects for which there is no evi-dence (1) or assuming that adsorption of the substrate canlead to unprecedented changes in the entropy of activation(2). Attempts to explain the specificity of enzyme reactionsare mostly based on Koshland's induced-fit theory (3), forwhich again there are no analogies.The active site of an enzyme is a cleft or hollow into which

its substrate fits closely. Adsorption of the substrate canthen take place only if all molecules of solvent (i.e., water)are squeezed out from between them. Thus, the enzyme re-action takes place in the absence of solvent, just like an anal-ogous reaction in the gas phase. Since gas-phase reactionsoften differ drastically from analogous reactions in solution,discussions of enzyme reactions clearly should be based onanalogies with the former. Yet all past and current mechanis-tic studies of enzyme reactions have assumed them to beanalogs of reactions in solution. This cannot but have led tomisconceptions and errors.Our purpose here is to show that the "problems" indicated

above become nonproblems if examined from this alterna-tive viewpoint.Activation Barriers in Solution: Autoactivated and"Solvactivated" Reactions

Recent work (4-10) indicates that reactions of ions with neu-tral molecules can be divided into two groups. In an autoac-tivated reaction, the reactants combine exothermically in thegas phase to form a charge-dipole adduct, separated by a

conventional activation barrier from the products. The SN2

reaction is an example, the corresponding minimum energyreaction path (MERP) in the gas phase being (4-7) of thetype indicated in Fig. 1, path a. In the gas phase, where theenergy liberated in forming the initial adduct cannot be rap-idly dissipated, the reaction may take place without activa-tion by a "hot molecule" mechanism, as indicated by thedotted line in Fig. 1, path a. In solution, the initial gain inenergy is eliminated (7, 8) leaving a conventional activationbarrier (Fig. 1, path b).Gas-phase reactions of the second type follow (7, 9, 10)

the course indicated by the schematic MERP labeled c inFig. 1. The reactants combine exothermically and withoutactivation to form a stable adduct. Any activation barrier tosuch a solvactivated reaction in solution must then be dueentirely to hindrance by the solvent, in particular to the factthat solvent must be removed from the ion before the otherreactant can approach. The transition state then correspondsto a solvated ion-dipole complex, little or no change in bond-ing having taken place. Since the adduct is larger than thereactant ion, its solvation energy should be correspondinglysmaller, so it is likely to become a high-energy intermediate,the MERP being as indicated by path d in Fig. 1.

Calculations and experiment (see ref. 9) suggest that sub-stitution at carbonyl carbon by anions, in particular hydroly-sis and alcoholysis of esters and amides by hydroxide or al-koxide ions, follow this pattern. Other reactions that seem tobe solvactivated are the SN2' reaction (7) and nucleophilicsubstitution on silicon (10).

Since adsorption of the substrate in the active site of anenzyme leads to total exclusion of solvent (water) from be-tween them, any subsequent reaction between them takesplace in the absence of solvent-i.e., as it would in the gasphase. The only difference is that any energy liberated in theinitial association can be rapidly dissipated, so "hot mole-cule" processes are no longer possible. Autoactivated reac-tions consequently encounter activation barriers analogousto those in solution. However, a solvactivated reaction willtake place without activation. Since the activation energiesfor the hydrolyses of amides by water, in the absence of ac-ids or bases, are usually >>100 kJ/mol, removal of the acti-vation barrier would be expected to increase the rate by atleast 20 orders of magnitude. This is more than enough toaccount for the whole of the acceleration observed in thehydrolysis of appropriate peptides by peptidases such aschymotrypsin.There are, however, logistic problems to be overcome in

implementing this scheme. How is the necessary anioniccenter generated in the active site of the enzyme, given thatreactions of this kind normally take place in neutral solution?And second, if such an anionic center were present, wouldnot its desolvation require as much energy as desolvation ofthe anion in a corresponding reaction in solution?

Abbreviation: MERP, minimum energy reaction path.*Present address: U.S. Air Force Academy, Colorado Springs, CO80840.

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2226 Biochemistry: Dewar and Storch

(b)Y RX

cW YRYRX (b)0~~~~~

X-+MI

(d)

Reaction Coordinate

FIG. 1. Schematic minimum energy reaction paths for ion-mole-cule reactions: paths a and b, an SN2 reaction; paths c, d, and b, a

solvactivated reaction; paths a and c, reactions in the gas phase;paths b and d, reactions in solution. The dotted line in path a indi-cates the course of a "hot molecule" reaction.

The Mechanism of Catalysis by Chymotrypsin

The structure of chymotrypsin and of its active site and thereactions involved in the catalytic hydrolysis of peptides byit seem to have been established (11, 12). The hydrolysistakes place in four steps, each involving the addition of an

anion (X-) to the carbonyl group of a carboxylic function inthe substrate (RCOY) or the reverse of such a process:

RX- + RCOY ;± X-C-0- [1]

Y

The relevant anions are generated by proton transfer fromHX to the carboxylate group of Asp-102 via the imidazolering of His-57 ("charge relay mechanism"):

(Asp)COO- H-N N H-X +± (Asp)COOH N NH X-. [2]

[Recent criticisms (13, 14) of this mechanism are refuted be-low.] The groups involved in each of the four steps (A-D) areas follows, the arrow indicating the direction in which Eqs. 1and 2 operate:

A(--) X = (Ser-195); Y = NHR'

B(+-) X = NHR'; Y = (Ser-195)

C(-*) X = HO; Y = (Ser-195)O

D(+-) X = (Ser-195); Y = HO.

RCOY is the peptide (RCONHR') in step A, the Ser-195 es-

ter ofRCOOH in steps B and C, and the acid (RCOOH) itselfin step D. HX is Ser-195 in A and D, the amine (R'NH2)derived from the peptide in C, and water in D. The reactionof Eq. 1 takes place in steps A and D from left to right and insteps B and D from right to left.Thermochemical data (15, 16) indicate that hydrolyses of

simple amides in the gas phase are weakly endothermic-e.g.,

HCONH2 + H20 -* HCOOH + CH3NH2

AH, 2.9 kJ/mole

CH3CONH2 + H20 -O CH3COOH + NH3AH, 8.4 kJ/mole

CH3CONHBu + H20 -* HCOOH + C4H9NH2AH, 22.4 kJ/mole

HCONMe2 + H20 -O HCOOH + HNMe2AH, 36.0 UJ/mole.

Water has little effect on the overall heat of reaction, thehydrolyses of peptides in aqueous solution being close tothermoneutral (17). Any catalytic process must be revers-ible. Under suitable conditions chymotrypsin can indeed cat-alyze the formation of amides from amines and acids (17).Peptides are hydrolyzed under biological conditions becauseof the gain in entropy, one molecule of reactant (peptide)being converted into two of products.

Since the adsorbed substrate is held firmly in place in theactive site with the reacting groups appropriately oriented,the reaction is effectively unimolecular, so the correspond-ing Arrhenius preexponential factor should be =1014. Sincethe corresponding first-order rate constants seem to be ca.102 (11, 12), the overall activation energy is ca. 60 kJ/mol.Our object is to show that, in the absence of solvent, the

four reactions (A-D) can take place fast enough to accountfor the rates of chymotrypsin-catalyzed cleavage of pep-tides, without any need to postulate additional factors. Theeffect of the enzyme could then be attributed wholly to ex-clusion of solvent. Since the hydrolysis takes place in foursteps and is almost thermoneutral, the activation energy ofeach step must be _60 kJ/mol, and none must be stronglyendothermic.No thermochemical data are available for tetrahedral ad-

ducts from carboxyl derivatives. We are forced to estimatethem theoretically. Since no current procedures can be ap-plied to enzymes, the calculations are confined to modelssimulating their active sites. Use of any adequate ab initioprocedure, even in this connection, would moreover bewholly impracticable. The only practicable models of ade-quate performance are MINDO/3 (18), MNDO (19), andAM1 (20), which give (21, 22) results comparable with thosefrom good ab initio ones at less than 1/1000th the cost incomputing time. Here AM1 was the obvious choice becauseit alone reproduces hydrogen bonds in a reasonably satisfac-tory manner (20).A proper model of chymotrypsin would include not only

the groups directly involved in the reaction but also adjacentpolar groups that can interact with them, either by hydrogenbonding or electrostatically. Calculations based on such amodel would represent a major undertaking, even usingAMi. Indeed, even the systems indicated in Eq. 1 would taxour current computer facilities. In this preliminary investiga-tion, we studied only simple models of the unit processesinvolved. More detailed calculations are planned, and a de-tailed account of those summarized below will be presentedelsewhere.

Since the intervening imidazole ring remains virtually un-changed during the charge relay, it can be ignored. Formicacid seemed a suitable model for aspartic acid since theirpKas (23) are similar (3.75 vs. 3.86). Methanol or ethanol wasused as a model for serine; N-methylformamide or N-methyl-acetamide, for the substrate; methylamine, for the amineformed by its cleavage; and methyl formate or methyl ace-tate, for the intermediate serine ester. The results quotedhere were for the combination methanol/N-methylforma-mide/methylamine/methyl formate. The results for otherchoices differed only by a few kilojoules per mole. All geom-etries were fully optimized. Calculations were carried out byusing the MOPAC program, available from the QuantumChemistry Program Exchange.

Proc. NatL Acad Sci. USA 82 (1985)

Proc. NatL. Acad. Sci. USA 82 (1985) 2227

Since the charge relay mechanism involves transfer of aproton from an alcohol to a carboxylate ion, it is naturallyvery endothermic. The calculated [observed (15, 16)] heat ofreaction for our model system is 128.4 (142.3) kJ/mol. Theactive site of chymotrypsin is therefore initially neutral. Thesubstrate can be adsorbed, and water eliminated, withouthindrance because the active site of chymotrypsin carries noionic charges. The anionic nucleophile is generated only af-ter the substrate has been adsorbed, in response to the de-mand of the latter for anions. Desorption of the productslikewise takes place without hindrance because the negativecharge is withdrawn to the aspartate group in chymotrypsin,leaving the active site neutral, before desorption takes place.This explains how the enzyme overcomes the difficulty not-ed earlier. The anionic center in the active site is developedonly after the substrate has been adsorbed.The heats of reaction (kJ/mol) calculated (AM1) for our

models of the individual steps were as follows: A, 50.2; B,-37.7; C, -31.4; D, 7.5.The values shown above are of course only approximate,

having been derived by an approximate method from a high-ly simplified model. The differences between the calculatedheats of reaction are nevertheless large enough to make itlikely that the first step (A) is the most endothermic and,therefore, rate determining. The experimental evidence sug-gests that this is indeed the case (24). It is the most endother-mic because nucleophilic addition to the carbonyl group inan amide is harder than analogous addition to a carboxylicacid or ester due to the greater resonance stabilization ofamides. Esters related to poor chymotrypsin substrates areoften hydrolyzed by chymotrypsin as fast as its true sub-strates (11, 12). Presumably the lesser endothermicity of thefirst step in the case of an ester compensates for retardationby odd molecules of water remaining in the active site.

In step A, the negative charge in the reactant resides onAsp-102, approximately half a unit on each oxygen, one oxy-gen being hydrogen-bonded to His-57 and the other to thehydroxyl of Ser-214 (11). In the product, the charge is con-centrated on the oxygen of the amide group undergoing hy-drolysis, which is hydrogen-bonded to the imino groups ofSer-195 and gly-193 (11). Therefore, hydrogen bondingshould not greatly alter the heat of reaction since the hydro-gen bonds in the reactants and products are formed by analo-gous species. The same argument also applies to the subse-quent steps.

Since the strength of a hydrogen bond is roughly propor-tional to the negative charge on the donor atom, the overallstabilization by hydrogen bonding should change linearlywith transfer of charge as the reaction proceeds. This repre-sents another basic difference from analogous ionic reac-tions in solution. Solvation of an ion in solution involves ori-entation of surrounding molecules of solvent by electrostaticinteractions with the ion. Dispersal of charge, in forming atransition state, leads to a decrease in such orientation and,hence, to a decrease in solvation energy. This is not the casein an enzyme reaction because the interacting groups in anenzyme-substrate adduct are fixed both in number and inorientation.The activation energy of each individual step (A-D) will

naturally be greater than its endothermicity. Moreover, eachis a multibond process, and multibond reactions are "forbid-den" (25)-i.e., they do not normally take place in a syn-chronous manner. However, migrations of hydrogen repre-sent an exception to the rule (25) because hydrogen is adeptat forming three-center bonds and also may undergo rapidmigration by tunneling. Hydrogen migration can occur,therefore, in concert with other processes without muchchange in the overall activation energy.The results reported here are consistent with our suggest-

ed mechanism. It seems reasonable to suppose that the rate

of the enzyme-catalyzed reaction is due entirely to the exclu-sion of solvent. Indeed, the efficiency with which chymo-trypsin catalyzes the hydrolysis of suitable peptides can beattributed to an ingenious expedient that enables the enzymeto carry out a gas-phase reaction in aqueous solution.

Role of Solvactivated Reactions in Enzyme Chemistry

Similar principles seem likely to hold generally in enzymechemistry, the extravagant rates of enzyme reactions beingdue simply to the fact that they take place in the absence ofsolvent, under conditions equivalent to the gas phase. If so,the basic steps in enzyme reactions must be solvactivatedbecause the activation barrier of an autoactivated processsurvives even in the absence of a solvent. The virtual ab-sence of pericyclic reactions and of the SN2 reaction fromlists of enzymatic processes is particularly significant in thisconnection. Almost the only exception is methylation, aprocess essential in biology and one that cannot easily bebrought about other than by the SN2 route. This reasoningleads to three hypotheses that may serve as general guides.HYPOTHESIS I. Enzyme reactions take place at the same

rate as analogous gas-phase reactions, apartfrom the possi-bility of "hot molecule" processes in the latter.HYPOTHESIS II. Reactions used by enzymes are usually

ones that involve no intermediate barriers in the gas phase.HYPOTHESIS III. Any charged group actively involved in

an enzyme reaction must be generated by some kind of relaymechanism after the substrate has been adsorbed. Chargedgroups can be present initially in an active site only if theycorrespond to charged groups of opposite sign in the sub-strate, the coulombic attraction between them being neededto squeeze out solvent.Some applications of these ideas are indicated below.Reactivation of Cholinesterase. Cholinesterase catalyzes

(11) the hydrolysis of acetylcholine by a charge-relay mecha-nism similar to that used by chymotrypsin. Nerve gases inac-tivate cholinesterase by phosphorylating the relevant serinemoiety, the resulting esters not being easily hydrolyzed. Theproblem is to explain how such relatively inert phosphoruscompounds can react with cholinesterase at an almost diffu-sion-controlled rate and yet form esters that are again rela-tively inert. Furthermore, the only nucleophiles that do reactrapidly with the serine esters, thus regenerating the enzyme,are oximes, compounds which do not usually show unusualreactivity as nucleophiles.The reactions of nerve gases with anions must clearly be

solvactivated. This is why they react so rapidly with cholin-esterase, provided of course that their molecules are of theright shape. Since they are only weakly polar, the solvactiva-tion barriers to their reactions with anions in solution will belarge (11, 12). This is why their reactions with nucleophilesin solution are so slow. However, once a nerve gas has react-ed with cholinesterase to form the corresponding serine es-ter, problems arise in hydrolyzing it. The hydrolysis, beingof SN2 type, requires the nucleophile to attack trans to theserine oxygen, as opposed to lateral attack involved in thecase of a carboxylic ester. Water, hydrogen-bonded to thehistidine, is unable to reach. To cleave the ester, one needs awater substitute-a molecule containing a crescent-shapedunit with an acidic hydrogen at one end to hydrogen bond tothe histidine and a nucleophilic group at the other end toattack phosphorus. Oximes meet these conditions perfectly.Note that according to this interpretation, it is the nitrogen ofthe oxime that attacks phosphorus.

Carboxypeptidase A. Senine peptidases cleave peptides bybase-catalyzed hydrolysis, using the same catalytic triad(Glu-His-Ser) as chymotrypsin. Carboxypeptidase A (2, 11,12) seems to act by acid-catalyzed hydrolysis, this beingneeded because it specifically cleaves the carboxyl-bearing

Biochemistry: Dewar and Storch

2228 Biochemistry: Dewar and Storch

terminal groups from peptides. The terminal carboxyl is ion-ized under biological conditions, and the resulting negativecharge would inhibit nucleophilic attack on the adjacent am-ide link.The active site of carboxypeptidase A contains a gluta-

mate ion (Glu-270) and a zinc ion, the latter being attached toanother glutamate residue (Glu-72), the imidazole rings oftwo histidine units (His-69 and His-196), and a molecule ofwater. Currently accepted mechanisms (2, 11, 12) for car-boxypeptidase A assume the zinc to act as a Lewis acid,coordinating to the amide carbonyl and, thus, promoting nu-cleophilic addition to it. The adduct formed by carboxypepti-dase A with glycyl-L-tyrosine, a poor substrate, has such astructure (26). The attacking nucleophile originally was as-sumed to be Glu-270, leading to a mixed anhydride-i.e.,

R, R,(Glu-270)COO- C=O-Zn+-- (Glu-270)COO-C-O-ZnH

R2NH R2NH

(Glu-270)COOCOR, + R2NH2 + Zn+. [3]

This mechanism would, however, violate Hypothesis III.Indeed, Breslow et al. (26) have shown that the anhydride isnot an intermediate. They suggest that the nucleophile is, infact, a molecule of water hydrogen-bonded to Glu-270, theacid being formed directly rather than by hydrolysis of ananhydride.

In the reverse reaction, where the enzyme catalyzes amideformation from an acid and an amine, this mechanism wouldinvolve displacement of the water bonded to zinc by the car-bonyl group of the acid. Since carboxylic acids are muchweaker bases than water and since the zinc carries a positivecharge, the displacement would be endothermic and proba-bly also would encounter a considerable solvactivation barri-er. It seems more likely that the catalyst is not the zinc itself,acting as a Lewis acid, but the molecule of water attached tozinc, acting as a protic acid:

O@H

(Glu-270)C- 0

O-/0ATH

R1

>C=O..HO-Zn+

R2NH H

-- (Glu-270)COOH HO-C-OH HOZn.

R2NH

If so, peptides that displace the water from zinc will not behydrolyzed, at least not by the "proper" route. This wouldexplain why glycyl-L-lysine is such a poor substrate. When itis adsorbed by carboxypeptidase A, the vital molecule of wa-ter is lost.Nobody seems as yet to have suggested this mechanism as

a possibility, probably because zinc in water behaves as a

base or a very weak acid. This, however, raises another ma-jor difference between gas phase and solution chemistry.The relative strengths of acids and bases in the gas phase are

often quite different from those in solution (27), and inorgan-ic hydroxides represent one of the more striking examples.Thus, silicic acid and aluminum hydroxide, both very weakacids in water, act as superacids in the gas phase, where theyconsequently serve as useful acid catalysts. It is, therefore,entirely possible that the molecule of water attached to Zn'in carboxypeptidase A may be strongly acidic, so long as

additional water is not present.Possible Role of Carbanions in Enzyme Reactions. Hydro-

carbons are also much stronger acids in the gas phase than in

solution (27). Thus, while toluene in solution is a weaker acidthan water by ±25 pKa units, in the gas phase it is stronger.Therefore, carbanions stabilized by substituents only a littlemore effective than phenyl could be formed as intermediatesin enzyme reactions.One of the major carbon-carbon bond-forming reactions

in biological systems is the Claisen condensation. However,it has been difficult to account for the formation of the nec-essary carbanions formed under enzymatic conditions. In-deed, Arnstadt (28) has claimed that free carbanions are notinvolved in enzymatic processes of this kind because no ex-change with 2H20 took place during them. Both of theseproblems disappear in the light of the present discussion.Since water is excluded from the active site during a "true"enzyme reaction, carbanions should be formed with corre-sponding ease, while the absence of water, of course, wouldmake exchange impossible.Carbanions also may play an unsuspected role in oxida-

tion-reduction processes by acting as hydride donors, quiteapart from their possible role as nucleophiles or equivalentsof organometallic species. Reductions by hydride transferare well recognized in reactions mediated by flavin or nico-tinamide coenzymes. Carbanions could be even more effec-tive. For example, they may play a role in the reduction ofdinitrogen (N2) by nitrogenase. Current mechanisms assumethis to take place by a series of steps, each involving reduc-tion by electron transfer, followed by protonation. The pos-sibility of reduction by hydride transfer, rather than electrontransfer, does not seem to have been considered.Redox Potentials. The differences between relative acid-

ities in solution and in the gas phase are due primarily to thedifferent energies of solvation of the (ionic) conjugate bases.Similar differences should occur in any reaction where aneutral molecule is converted to an anion, in particular elec-tron-transfer processes. Therefore, differences between re-dox potentials in solution and in the gas phase should parallelthose in the pKa of acids. Reductions involving the forma-tion of carbanions should take place, for example, muchmore easily in the gas phase (or during an enzyme reaction)than in solution. Therefore, attempts to assess electron-transfer mechanisms for enzyme reactions from convention-al redox potentials may prove to be misleading.

"Artificial Enzymes." The conclusions reached here are ofobvious relevance to attempts to synthesize "artificial en-zymes." If such a species is to bring about rate enhance-ments comparable with those due to enzymes, it is not suffi-cient for it to contain the necessary reactive groups in theright orientation. The "active site" in the artificial enzymealso must fit the substrate closely enough to ensure that ad-sorption of the latter can occur only if all solvent is extrudedfrom between them. If an ion is involved in the reaction, itmust also be formed only after the substrate has been ad-sorbed. Attempts to promote reactions in this way are,moreover, likely to prove abortive unless they are of the solv-activated type.

Implications Concerning Methodology

The arguments above have general implications concerningthe methodology to be followed in studying enzyme reac-tions.

(i) Studies of enzymes with their active sites full of waterhave no relevance to their reactions with substrates. In par-ticular, attempts (cf. ref. 13) to assess the possibility of pro-ton transfers from pKa measurements under such conditionsare meaningless because the relative strengths of acids are

altered radically by removal of solvent.(ii) Similar remarks apply to attempts to determine the

mechanisms of enzyme reactions from studies of analogousreactions of "poor substrates." The latter react more slowly

Proc. NatL Acad Sci. USA 82 (1985)

Proc. Natl. Acad. Sci. USA 82 (1985) 2229

simply because they do not fill the active site of the enzyme.Such reactions are indeed analogs of reactions in solutionand so bear no necessary relation to the true enzymatic pro-cesses.

(iii) The use of enzyme-inhibitor adducts as models of theenzyme-substrate combination is also unsatisfactory forsimilar reasons. Unless the inhibitor fills the active siteclosely, it will not be a good model for a real substrate. Thereis also the danger that the inhibitor may be an inhibitor sim-ply because it reacts with the active site in a manner differentfrom the substrate.

(iv) Any approach to enzyme reactions along the linessuggested here depends on the availability of informationconcerning the energetics of reactions in the complete ab-sence of solvent. Experimental data of this kind are scanty,partly because techniques have been developed only recent-ly for studying ion-molecule reactions in the gas phase andpartly because the demand for such information has beenlimited. Certainly no one has suggested that it could be ofmajor interest to biochemists.

(v) The arguments presented here suggest that theoreticalcalculations could* play a very effective role in enzymechemistry, given that such calculations normally refer to re-actions of isolated molecules. Calculations for effectivemodels of enzymes have seemed far out of reach in the pastbecause of the lack of a suitable theoretical procedure. Asindicated above, AM1 seems to meet this need. We hope itwill prove as useful in enzyme chemistry as MINDO/3 andMNDO have already done in studies of organic reactions.

This work was supported by the Air Force Office of ScientificResearch (Contract F49620-83-C-0024) and the Robert A. WelehFoundation (Grant F-126).

1. Lipscomb, W. N. (1982) Acc. Chem. Res. 15, 232-238.2. Jencks, W. P. (1982) J. Biol. Chem. 257, 10893-10907.3. Koshland, D. E., Jr. (1%3) Cold Spring Harbor Symp. Quant.

Biol. 28, 473-482.4. Olmstead, W. N. & Brauman, J. I. (1977) J. Am. Chem. Soc.

99, 4219-4228.5. Keil, F. & Ahlrichs, R. (1976) J. Am. Chem. Soc. 98, 4787-

4793.

6. Wolfe, S., Mitchell, D. J. & Schlegel, H. B. (1981) J. Am.Chem. Soc. 103, 7692-76%.

7. Dewar, M. J. S. & Carrion, F. (1984) J. Am. Chem. Soc. 106,3531-3539.

8. Chandrasekhar, J., Smith, S. F. & Jorgensen, W. L. (1984) J.Am. Chem. Soc. 106, 3049-3050.

9. Dewar, M. J. S. & Storch, D. M. (1984) J. Chem. Soc. Chem.Commun., in press.

10. Dewar, M. J. S. & Healy, E. (1982) Organometallics 1, 1705-1708.

11. Blackburn, S. (1976) Enzyme Structure and Function (Dekker,New York).

12. Ferscht, A. R. (1977) Enzyme Structure and Mechanism (Free-man, San Francisco).

13. Bachovchin, W. W. & Roberts, J. D. (1978) J. Am. Chem.Soc. 100, 8041-8047.

14. Kossiakoff, A. A. & Spencer, S. A. (1981) Biochemistry 20,6462-6474.

15. Cox, J. D. & Pilcher, G. (1970) Thermochemistry of Organicand Organometallic Compounds (Academic, New York).

16. Pedley, J. B. & Rylance, J. (1977) Sussex-N.P.L. ComputerAnalysed Thermochemical Data: Organic and OrganometallicCompounds (Sussex Univ., Brighton).

17. Fruton, J. S. (1982) Adv. Enzymol. 53, 239-307.18. Binkham, R. C., Dewar, M. J. S. & Lo, D. H. (1975) J. Am.

Chem. Soc. 97, 1285-3111.19. Dewar, M. J. S. & Thiel, W. (1977) J. Am. Chem. Soc. 99,

4899-4917.20. Dewar, M. J. S., Healy, E. F., Stewart, J. J. P. & Zoebisch,

E. G..(1985) 1; Am. Chem. Soc., in press.21. Dewar, M. J. S. & Ford, G. P. (1q79) J. Am. Chem. Soc. 101,

5558-5561.22. pewar, M. J. S. & Storch; D. (1985) J. Am. Chem. Soc., in

press.23. Weast, R. C., Astle, M. J. & Beyer, W. H., eds. (1984) CRC

Handbook of Chemistry.and Physics (CRC Press, West PalmBeach, FL), 54th Ed., D-166.

24. Dewar, M. J. S. (1984) J. Am. Chem. Soc. 106, 209-219.25. Hunkapiller, M. W., Forgac, M. D. & Richards, J. H. (1976)

Biochemistry 15, 5581.26. Breslow, R. & Wernick, D. (1976) J. Am. Chem. Soc. 98, 259-

261.27. Bowers, M. T., ed. (1979) Gas Phase Ion Chemistry (Academ-

ic, New York), Vol. 2.28. Arnstadt, K.-I., Schindbleck, G. & Lynen, F. (1975) Eur. J.

Biochem. 55, 561.

Biochemistry: Dewar and Storch