Proc. Natl. Acad. Sci. USA Vol. 82, pp. 2225-2229, April 1985 Biochemistry 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 active reaction is an example, the corresponding minimum energy 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 molecule" mechanism, as indicated by the lution. Therefore, current interpretations of enzyme reactions dotted line in Fig. 1, path a. In solution, the initial gain in in terms of solution chemistry are misguided. The large rates energy is eliminated (7, 8) leaving a conventional activation and specificity of enzyme reactions may be due simply to elimi- barrier (Fig. 1, path b). nation of the solvent. The cleavage of peptides by chymotryp- Gas-phase reactions of the second type follow (7, 9, 10) sin and carboxypeptidase 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 ion before the other orders of magnitude than other catalysts, while reactions of reactant can approach. The transition state then corresponds other analogous molecules 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- lead to unprecedented changes in the entropy of activation sis and alcoholysis of esters and amides by hydroxide or al- (2). Attempts to explain the specificity of enzyme reactions koxide ions, 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 active site 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 leads to total exclusion of solvent (water) 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 energies 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 hydrolysis of appropriate peptides by peptidases such as Activation Barriers in Solution: Autoactivated and chymotrypsin. "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 acids (17). I Peptides are hydrolyzed under biological conditions because of 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 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 activation energy 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 Catalysis 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 function 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 imidazole 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 hydrogen bonds in a reasonably satisfac- (Asp)COO- H-N N H-X +± (Asp)COOH N NH X-.