CRYOENZYMOLOGY OF CHYMOTRYPSIN - DETECTION OF INTERMEDIATES IN CATALYSIS OF A SPECIFIC ANILIDE SUBSTRATE

The reaction between chymotrypsin and N-acetyl-L-phenylalanine p-nitroanilide was studied at subzero temperatures in fluid aqueous dimethyl sulfoxide solvent. Following initiation of the reaction at temperatures as low as -90.degree. C, a series of 4 reactions prior to the normal rate-limiting step (acylation) was detected spectrophotometrically. Various experimental observations have led to the following interpretation of these reactions. Reaction 1 corresponds to the binding of substrate yielding the initial Michaelis complex. Reactions 2 and 3 are 2 pH-independent reactions, ascribed to substrate-induced changes in the positions of active-site groups. Reaction 4 is a pH-dependent reaction (pK = 5.9) which involves the imidazole of His-57 but which is not the formation of a tetrahedral intermediate, oxazolinone or acyl enzyme. The slowest detected step corresponded to the acylation reaction. No evidence for the accumulation of a tetrahedral intermediate was obtained. Spectral, kinetic and thermodynamic data for these reactions are presented, as is justification for the relevance of these findings to the reaction under physiological conditions. These results demonstrate the utility of subzero temperatures in enzyme mechanism studies, especially with regard to allowing the accumulation of intermediates which may be quite stable at appropriate values of pH and low temperature.