Mechanism of action of serine proteases: tetrahedral intermediate and concerted proton transfer.

Abstract

Stopped-flow spectrophotometry and proton inventory experiments have been used to define the reaction pathway for hydrolysis of a specific peptide substrate, Ac-L-Ala-L-Pro-L-Ala p-nitroanilide, by the serine proteases elastase and alpha-lytic protease. The stopped-flow studies reveal the existence and buildup of a tetrahedral adduct between the active site serine hydroxyl group and the sensitive carbonyl group of the substrate. The decomposition of this tetrahedral intermediate to the acyl enzyme and p-nitroaniline is the rate-limiting step for the hydrolytic reaction. The proton inventory data suggest the simultaneous transfer of two protons (presumably from the catalytic carboxyl of Asp-102 to N pi of the catalytic imidazole of His-57 and from N pi of the imidazole to the anilide NH) in the transition state leading to breakdown of the tetrahedral complex. That these proton transfers occur in a concerted, rather than stepwise, process attests to the ability of enzymes to lower the enthalpy of activation most effectively when the precise alignment of a highly specific substrate and catalytic groups minimizes the entropy of activation.