MULTIDIELECTRIC DESCRIPTION OF ELECTROSTATIC ENVIRONMENT SURROUNDING A BOUND SUBSTRATE IN ENZYMATIC SYSTEMS

The molecular mechanism of serine protease catalysis was investigated using a reaction field theory combined with molecular orbital calculat ion. The active site of the protein was represented as a multidielectr ic system. The region of the so-called oxyanion hole Was characterized as a microscopic domain with a high dielectric constant and the other region as a relatively low polarizable medium. A representative subst rate, methyl formate, is embeded in such a pseudo-protein matrix with its carbonyl bond directing toward the oxyanion hole mimic. The energy profile was obtained for alkaline hydrolysis of the substrate, using a computational methodology recently developed by us. It was found tha t the rate-limiting step of the reaction is the demethoxylation from t he well-known tetrahedral intermediate, and its energy profile sensiti vely depends on the electrostatic nature of the surrounding protein ma trix. In particular, the reaction field generated from the oxyanion ho le contributes to electrostatically stabilizing the tetrahedral interm ediate. However, the most important finding is that the transition sta te is destabilized by the presence of the oxyanion hole, leading to an increase in the activation energy. This result may be inconsistent wi th the conventional picture of serine protease hydrolysis.