Solvation, Reorganization Energy, and Biological Catalysis

Abstract

transition state analog. The question of how enzymes greatly enhance the rate of reactions has been discussed for years but remains a vigorously debated issue. Rapid progress has been made on the mechanism of individual enzymes by a combination of kinetic, chemical, and structural approaches. The push and pull of electrons and the resulting bond changes are well understood for many enzymes. However, the larger question of general features that enzymes use to produce rate accelerations of 108–1015 has remained a contentious issue. We believe that such rate accelerations can be readily explained by reasonable physical principles (1Cannon W.R. Singleton S.F. Benkovic S.J. Nat. Struct. Biol. 1996; 3: 821-833Crossref PubMed Scopus (134) Google Scholar). Though much of this understanding stems from research done in the 1970s and 1980s, these insights are often underappreciated or even completely neglected when examining enzymic rate accelerations. Perhaps this is because of our intense focus on enzymes themselves and relative neglect of the reference solution reactions to which they are compared, consequentially, resulting in a mechanistic tunnel vision. Drawing on an earlier analysis (1Cannon W.R. Singleton S.F. Benkovic S.J. Nat. Struct. Biol. 1996; 3: 821-833Crossref PubMed Scopus (134) Google Scholar) and work cited herein, we wish to examine a popular model for comparing the rates of catalyzed and uncatalyzed reactions, that is, the thermodynamic cycle as set up in Scheme 1, which compares a transformation of a reactant (S) in solution through its transition state (S‡) with the same reaction catalyzed by an enzyme (E) from its ground state (ES) to its transition state (ES‡). E+S →knon E+S‡K=1↑↑KTS E+S ←Km ES →kcat ES‡SCHEME IThe solution reaction is characterized by the reaction ratek non, and the enzyme-catalyzed reaction is characterized by the Michaelis-Menten constantsk cat and Km . An apparent equilibrium constant for the dissociation of the transition state of the reactant from the enzyme can be obtained using transition state theory (2Kurz J.L. J. Am. Chem. Soc. 1963; 85: 987-991Crossref Scopus (128) Google Scholar), K TS =((k cat /Km)/k non)−1. The value of K TS is generally significantly smaller than the dissociation constant for the substrate from the enzyme, KS, leading to a large and favorable free energy of binding for the enzyme and S‡ (3Wolfenden R. Acc. Chem. Res. 1972; 5: 10-18Crossref Scopus (546) Google Scholar, 4Lienhard G.E. Science. 1973; 180: 149-154Crossref PubMed Scopus (417) Google Scholar, 5Radzicka A. Wolfenden R. Science. 1995; 267: 90-93Crossref PubMed Scopus (741) Google Scholar). As a result of the small value of K TS, analogs of the reactant in its transition state geometry (transition state analog (TSA)1) have been sought as enzyme inhibitors. In addition, TSAs and the scenario of Scheme 1 have been used as the foundation for the design of catalysts from antibodies, in which immunization against a TSA is used to elicit the formation of antibodies that strongly bind the TSA. The thermodynamic cycle set up in Scheme 1 is useful as a model for understanding many aspects of rate accelerations. However, as in all models of complex phenomena, its limitations must also be kept in mind. Without conjecture, all we can say from the analysis of the thermodynamic cycle is that, if the virtual binding of the reactant in its transition state geometry were physically able to occur, the process would be very favorable in terms of free energy. The difference in the apparent free energy of binding of S‡ to the enzyme relative to S may result from two scenarios. In the first scenario, significantly stronger interactions may exist between the enzyme and the reactant in its transition state configuration than in the enzymic ground state configuration. In this case, as the enzymic reaction proceeds along the lower path in Scheme 1, the binding of the reactant to the enzyme increases significantly. As an example, it has been proposed that short, strong hydrogen bonds could account for much of the “missing” energy required for the differential stabilization of the ES‡ complex compared with the ground state ES complex (6Cleland W.W. Kreevoy M.M. Science. 1994; 264: 1887-1890Crossref PubMed Scopus (1048) Google Scholar) (see the first minireview in this series by Cleland et al. (48Cleland W.W. Frey P.A. Gerlt J.A. J. Biol. Chem. 1998; 273: 25529-25532Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar) for details). The second possibility is that the free energy of interaction between the solvent environment and S‡ is much less favorable than between the solvent and S. Then, as the solution reaction proceeds toward the transition state along the upper path in Scheme 1, the free energy change includes a large and significant contribution from solvation. Although it is clear that the correct answer is a combination of the two scenarios, it is important for catalyst design purposes to de