Solvation, Reorganization Energy, and Biological Catalysis

Abstract

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 10 8 -10 15 has remained a contentious issue. We believe that such rate accelerations can be readily explained by reasonable physical principles (1). 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. Transition State Theory and Thermodynamic CyclesDrawing on an earlier analysis (1) 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 ‡ ).The solution reaction is characterized by the reaction rate k non , and the enzyme-catalyzed reaction is characterized by the Michaelis-Menten constants k cat and K m . An apparent equilibrium constant for the dissociation of the transition state of the reactant from the enzyme can be obtained using transition state theory (2),Ϫ1 . The value of K TS is generally significantly smaller than the dissociation constant for the substrate from the enzyme, K S , leading to a large and favorable free energy of binding for the enzyme and S ‡ (3-5). 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. Can We Do Better?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, ...

H-tunneling in the Multiple H-transfers of the Catalytic Cycle of Morphinone Reductase and in the Reductive Half-reaction of the Homologous Pentaerythritol Tetranitrate Reductase

“…Our previous studies with wild-type trimethylamine dehydrogenase and the Y169F mutant (48), and reactions of methylamine dehydrogenase with methylamine and ethanolamine (44,47) have demonstrated both temperature-independent and temperature-dependent KIEs, suggesting that tunneling can proceed in reactions dominated either by "gated" or "Frank-Condon" dynamics in both enzymes. 3 Similar explanations have been advanced to explain temperature-dependent KIEs in wild-type and mutant forms of soybean lipoxygenase-1 (24). The experimental evidence with these enzymes is therefore consistent with H-transfer by environmentally coupled hydrogen tunneling and can be modeled satisfactorily using the theoretical framework of Kuznetsov and Ulstrup (19).…”

“…Our quest to understand the physical basis of this catalytic power is challenging and has involved sustained and intensive research efforts by many workers in the physical and life sciences (for recent reviews see Refs. [2][3][4][5]. Recent years have witnessed new activity in this area and extended our theoretical understanding beyond the shortcomings of transition state theory (6) to include roles for protein "motion" (7,8), low barrier hydrogen bonds (e.g.…”

Detection of Large pK Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes

“…Each sample contained 15 mg/ml protein and saturating amounts of steroid in a solution composed of 0.45 ml of phosphate buffer (10 mM, pH 7.5 or 8.5) and 0.05 ml of dimethyl sulfoxide-d 6 . Jump-and-return pulse sequence (17) was employed to suppress the water signal.…”

Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

“…It has also been suggested that dynamic excursions of active site architecture on the picosecond timescale create variable electronic interactions between enzyme and reactants, which induce catalytic barrier crossing (7,8,10,17,19). In an alternative theory of enzyme catalysis, it has been proposed that electrostatic forces, due to preorganized active site dipoles and charges, primarily drive the formation of the transition state during enzymatic catalysis, and dynamic motion of the active site residues are unimportant or even deleterious (23)(24)(25)(26)(27)(28)56).…”

“…Theoretical studies have suggested that fast vibrations in enzymes might generate transition state conformations conducive to the chemical reaction (7,8,(14)(15)(16)(17)(18)(19)(20)(21), a familiar concept for reactions in ordinary solvents (22). In an alternative viewpoint, preorganization effects have been suggested to be a major contributing factor to enzyme catalysis (23)(24)(25)(26)(27)(28). It has been postulated that enzymes have partially oriented dipoles of polar and charged groups in the active site that interact with electrostatic features present in the catalytic transition state more favorably than water can.…”