DIFFERENTIAL ACTIVATION OF ENDOPEPTIDASE EC-3.4.24.15 TOWARD NATURAL AND SYNTHETIC SUBSTRATES BY METAL-IONS

The activity of endopeptidase EC 3.4.24.15 (thimet oligopeptidase, EP 24.15), as measured by cleavage of a quenched fluorescent substrate, 7 -methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-D-Lys (2,4-dinitrophenyl), w as increased 2-3 fold by the addition of 1 mM Mn2+ or of 10 mM Ca2+. T he inhibitory capability of a specific EP. 24.15 inhibitor, rboxy-3-ph enylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, was also increased at similar concentrations of these metal ions. However, the hydrolysis of natura lly-occurring peptides, thought to be the physiological substrates for EP 24.15, was not affected by either Mn2+ or Ca2+. These results sugg est that the binding of synthetic analogs to the enzyme may differ sig nificantly from the binding, and thus hydrolysis, of natural peptide s ubstrates and caution against drawing conclusions about substrate inte ractions with the active site from data obtained with modified peptide ligands. (C) 1996 Academic Press, Inc.