BIFUNCTIONAL PEPTIDYLGLYCINE ALPHA-AMIDATING ENZYME REQUIRES 2 COPPERATOMS FOR MAXIMUM ACTIVITY

The conversion of C-terminal glycine-extended peptides to C-terminal a lpha-amidated peptides occurs in two distinct reactions, both of which are catalyzed by bifunctional peptidylglycine alpha-amidating enzyme. The first step is the alpha-hydroxylation of the C-terminal glycine r esidue and the second step is the dealkylation of the alpha-hydroxygly cine-extended peptide to the ar-amidated peptide and glyoxylate. We sh ow that the bifunctional enzyme requires 1.9 +/- 0.2 mol of copper/mol of enzyme for maximal dansyl-Tyr-Lys-Gly amidation activity under the conditions of high enzyme concentration (similar to 80 mu M) required to measure initial rates for this poor substrate. The enzyme, as puri fied, contains a substoichiometric amount of copper and has only trace levels of amidation activity. Addition of exogenous Cu(II) ions stimu lates amidation activity similar to 3000-fold at the optimum copper st oichiometry and the enzyme is then inhibited by excess Cu(II). No stim ulation of amidation activity is observed upon the addition of the fol lowing divalent metal ions: Mn(II), Fe(II), NI(II), Cd(II), and the ox ovanadium cation, VO(II). The enzyme-catalyzed dealkylation of alpha-h ydroxyhippuric acid to benzamide shows no dependence on copper, indica ting that the copper dependence of the amidation reaction must be attr ibuted to a copper dependence in peptide alpha-hydroxylation. (C) 1994 Academic Press, Inc.