Arazoformyl dipeptide substrates for thermolysin. Confirmation of a reverse protonation catalytic mechanism.

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 Kcat and Km 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 (pKa of 5, seen in Km) 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 (pKa of 8, seen in Kcat), 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 pKa 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.