Arazoformyl Dipeptide Substrates for Thermolysin. Confirmation of a Reverse Protonation Catalytic Mechanism

Abstract

Cleavage by thermolysin of N-(4-methoxyphenylazoformyl)-l-leucyl-l-leucine plus some congeneric peptides provides a highly sensitive new kinetic assay for proteolytic activity. The pH dependence of Michaelis−Menten parameters k_cat and K_m establishes kinetically a reverse protonation catalytic mechanism for this metalloprotease [Mock, W. L., & Aksamawati, M. (1994) Biochem. J. 302, 57−68]. An acidified water molecule (pK_a of 5, seen in K_m) becomes displaced by substrate carboxamide from the hypercationic Zn²⁺ of the enzyme, yielding potent Lewis acid activation of the peptide linkage for subsequent hydrolysis. Conversion to product is induced by the side chain of enzymic residue His 231 (pK_a of 8, seen in k_cat), which provides general base catalysis for addition of H₂O to the zinc-activated scissile carboxamide of the bound substrate. A previously described “superactivation” through chemical modification of the enzyme with acetylphenylalanyl-N-hydroxysuccinimide is nonexistent in the case of the new substrates, which indicates that their binding to thermolysin is largely productive, unlike normal peptides. Correct assignment of kinetically observed pK_a values to active site residues, along with recognition of a predominantly nonproductive binding mode for ordinary substrates and thermolysin, forces reinterpretation of previous mechanistic formulations for the enzyme.