Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases.

Abstract

Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases being unreactive toward other classes of proteinases. Variation of the peptide portion of the reagents has provided affinity labels for the selective inactivation of the thiol endopeptidase cathepsin B, streptococcal proteinase, and clostripain and for the aminodipeptidase cathepsin C. Comparison of rates of inactivation of cathepsin B and streptococcal proteinase revealed a similarity in specificity, both enzymes showing a preference for a phenylalanine residue in the penultimate position of the peptide portion of the reagent. Reagents not satisfying the specificity of a particular thiol proteinase either did not inactivate or did so only very slowly. In some cases, the reagents bound to the enzyme in a manner unproductive for alkylation but productive for substrate behavior leading to destruction of the inhibitor by cleavage of the peptide portion. Thus, benzyloxycarbonyl-Phe-Gly-Phe diazomethyl ketone was rapidly cleaved by cathepsin B into benzyloxycarbonyl-Phe-Gly and phenylalanine diazomethyl ketone. Clostripain, an enzyme of trypsin-like specificity, was selectively inactivated by benzyloxycarbpnyl-Lys diazomethyl ketone at micromolar concentrations, but was inactivated extremely slowly by 2.5 x 10/sup -4/ M benzyloxycarbonyl-Phe-Ala diazomethyl ketone, a very effective inactivated by Gly-Phe diazomethyl ketone in the range of 10/sup -7/ to 10/sup -8/ M.more » By comparison, peptidyl diazomethyl ketones with blocked NH/sub 2/ terminals were considerably less effective. Peptidyl diazomethyl ketones are unreactive with such thiols as mercaptoethanol and glutathione.« less