How Much Do Enzymes Really Gain by Restraining Their Reacting Fragments? A. Shurki, † M. S ˇ trajbl, † J. Villa ` , ‡ and A. Warshel* ,† Contribution from the Department of Chemistry, UniVersity of Southern California, Los Angeles, California 90089-1062 Received September 24, 2001 Abstract: The steric effect, exerted by enzymes on their reacting substrates, has been considered as a major factor in enzyme catalysis. In particular, it has been proposed that enzymes catalyze their reactions by pushing their reacting fragments to a catalytic configuration which is sometimes called near attack configuration (NAC). This work uses computer simulation approaches to determine the relative importance of the steric contribution to enzyme catalysis. The steric proposal is expressed in terms of well defined thermodynamic cycles that compare the reaction in the enzyme to the corresponding reaction in water. The S N2 reaction of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, which was used in previous studies to support the strain concept is chosen as a test case for this proposal. The empirical valence bond (EVB) method provides the reaction potential surfaces in our studies. The reliability and efficiency of this method make it possible to obtain stable results for the steric free energy. Two independent strategies are used to evaluate the actual magnitude of the steric effect. The first applies restraints on the substrate coordinates in water in a way that mimics the steric effect of the protein active site. These restraints are then released and the free energy associated with the release process provides the desired estimate of the steric effect. The second approach eliminates the electrostatic interactions between the substrate and the surrounding in the enzyme and in water, and compares the corresponding reaction profiles. The difference between the resulting profiles provides a direct estimate of the nonelectrostatic contribution to catalysis and the corresponding steric effect. It is found that the nonelectrostatic contribution is about -0.7 kcal/mol while the full “apparent steric contribution” is about -2.2 kcal/mol. The apparent steric effect in- cludes about -1.5 kcal/mol electrostatic contribution. The total electrostatic contribution is found to account for almost all the observed catalytic effect (∼-6.1 kcal/mol of the -6.8 calculated total catalytic effect). Thus, it is concluded that the steric effect is not the major source of the catalytic power of haloalkane dehalogenase. Furthermore, it is found that the largest component of the apparent steric effect is associated with the solvent reorganization energy. This solvent-induced effect is quite different from the traditional picture of balance between the repulsive interaction of the reactive fragments and the steric force of the protein. 1. Introduction The molecular origin of enzyme catalysis is a problem of major fundamental and practical importance. Biochemical and structural studies have provided the groundwork for tackling this problem (e.g., ref 1). Yet, discrimination between dif- ferent proposals for the source of enzyme catalysis still re- quires quantitative structure-function correlation studies (e.g., ref 2). Furthermore, even the advance of computer simula- tion approaches for studies of enzymatic reactions (for re- cent review, see ref 3) has not yet provided a consensus in the field. Here we address the proposal that enzymes exert on their re- actants some form of reactants state (RS) strain, and thus “compress” or “mold” the reacting fragments to a configuration that resembles the transition state, TS, (e.g., refs 4-9). Un- fortunately, the processes that have been studied do not appear to be directly relevant to the strain proposal. Moliner et al. 8 for example, compared the QM/MM structure of the TS in the * To whom correspondence should be addressed. E-mail: warshel@ usc.edu. † University of Southern California. ‡ Present address: Grup de Recerca en Informa `tica Biome `dica IMIM/ UPF C/Doctor Aiguader, 80 08003 Barcelona, Spain. (1) Fersht, A. Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding, 2nd ed.; W. H. Freeman and Company: New York, 1999. (2) Warshel, A. Computer Modeling of Chemical Reactions in Enzymes and Solutions; John Wiley & Sons: New York, 1991. (3) Villa `, J.; Warshel, A. J. Phys. Chem. B 2001, 105, 7887-7907. (4) Ford, L. O.; Johnson, L. N.; Machin, P. A.; Phillips, D. C.; Tjian, R. J. Mol. Biol. 1974, 88, 349-371. (5) Khanjin, N. A.; Snyder, J. P.; Menger, F. M. J. Am. Chem. Soc. 1999, 121, 11831-11846. (6) Tapia, O.; Andre ´s, J.; Safont, V. S. J. Chem. Soc., Faraday Trans. 1994, 90, 2365-2374. (7) Castillo, R.; Andre ´s, J.; Moliner, V. J. Am. Chem. Soc. 1999, 121, 12140- 12147. (8) Moliner, V.; Andre ´s, J.; Oliva, M.; Safont, V. S.; Tapia, O. Theor. Chem. Acc. 1999, 101, 228-233. (9) Martı ´, S.; Andre ´s, J.; Moliner, V.; Silla, E.; Tun ˜o ´n, I.; Bertra ´n, J. J. Phys. Chem. B 2000, 104, 11308-11315. Published on Web 03/20/2002 10.1021/ja012230z CCC: $22.00 © 2002 American Chemical Society J. AM. CHEM. SOC. 2002, 124, 4097-4107 9 4097