R. Kulathila et al., BIFUNCTIONAL PEPTIDYLGLYCINE ALPHA-AMIDATING ENZYME REQUIRES 2 COPPERATOMS FOR MAXIMUM ACTIVITY, Archives of biochemistry and biophysics, 311(1), 1994, pp. 191-195
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.