Kinetic and stereochemical studies on novel inactivators of C-terminal amidation

C-terminal amidation, a required post-translational modification for the bi oactivation of many neuropeptides, entails sequential enzymic action by pep tidylglycine il-mono-oxygenase (PAM, EC 1.14. 17.3) and peptidylamidoglycol ate lyase (PGL, EC 4.3.2.5). Here we introduce novel compounds in which an olefinic functionality is incorporated into peptide analogues as the most p otent turnover-dependent inactivators of PAM. Kinetic parameters for PAM in activation by 4-oxo-5-acetamido-6-phenyl-hex-2-enoic acid and 4-oxo-5-aceta mido-6-(2-thienyl)-hex-2-enoic acid were obtained by using both the convent ional dilution assay method and the more complex progress curve method. The results obtained from the progress curve method establish that these compo unds exhibit the kinetic characteristics of pure competitive inactivators ( i.e. no ESI complex forms during inactivation). On the basis of k(inaet)/K- i values, 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid is almost two or ders of magnitude more potent than benzoylacrylate, a chemically analogous olefinic inactivator that lacks the peptide moiety. Stereochemical studies established that PAM inactivation by 4-oxo-5-acetamido-6-(2-thienyl)-hex-2- enoic acid is stereo-specific with respect to the moiety at the P-2 positio n, which is consistent with previous results with substrates and reversible inhibitors. In contrast, 2,4-dioxo-5-acetamido-6-phenylhexanoic acid, whic h is a competitive inhibitor with respect to ascorbate, exhibits a low degr ee of stereospecificity in binding to the ascorbate sites of both PAM and d opamine-beta-hydroxylase.