Mechanistic studies of carboxypeptidase Y. Kinetic detection of an acyl-enzyme intermediate in trimethylacetate esterase action

Carboxypeptidase Y (protease C) from Saccharomyces cerevisiae possesses esterase activity toward the nonspecific substrates, aryl trimethylacetates. By means of stopped-flow spectrophotometry, the burst kinetics observed for 4-nitrophenyl trimethylacetate hydrolysis catalyzed by carboxypeptidase Y have been studied in detail. The results are quantitatively consistent with the intermediacy of a physical complex between enzyme and substrate followed by an acyl enzyme, trimethylacetylcarboxypeptidase Y. Acylation rate constants (k₂/Ks) show a bell-shaped pH dependence on ionizations of pKapp = 5.94 and 7.92. Deacylation rate constants (k₃) show a sigmoidal dependence on an ionization of a group of pKapp = 5.1. This group is noncrucial as the rate constant in acidic media (0.99 × 10⁻² s⁻¹) is 41% of the maximal rate constant in alkaline media (2.42 × 10⁻² s⁻¹). In agreement with the acyl-enzyme hypothesis, kcat. values for a series of aryl-substituted trimethylacetates are independent of leaving group. The mechanism of action and site of acylation of carboxypeptidase Y are discussed in terms of the extent of catalysis in both acylation and deacylation and the natures of the catalytic groups involved.