title: Further characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary.
abstract: The ability of purified bovine neurointermediate pituitary peptidyl glycine alpha-amidating monooxygenase to catalyze the conversion of peptide substrates (D-Tyr-X-Gly) into amidated product peptides (D-Tyr-X-NH2) was evaluated. The pH optimum of the reaction was pH 8.5 when X was Val, Trp, or Pro, but 5.5 to 6.0 when X was Glu. Similar maximum velocity (Vmax) values were obtained for the Val, Trp, and Pro substrates while the Glu substrate had a substantially higher Vmax. The Michaelis-Menten constant (Km) of the enzyme for the peptide substrate increased in the order Trp less than Val less than Pro much less than Glu. Increasing levels of ascorbate brought about parallel increases in Km and Vmax, suggesting the presence of an irreversible step separating the interaction of the enzyme with the two substrates. The effect of copper on enzyme activity was dependent on the peptide substrate and the reaction pH. With the Val substrate, exogenous copper was required for optimal activity; no other metal ion tested could substitute for copper. With the Glu substrate, exogenous copper was not required for optimal activity; however, diethyldithiocarbamate, a copper chelator, inhibited activity and only copper could reverse this inhibitory effect. The ability of various cofactors to stimulate alpha-amidating monooxygenase activity was also dependent on assay conditions. With the Val or Glu substrate in the presence of exogenous copper, a variety of cofactors in addition to ascorbate were capable of supporting activity. With the Glu substrate in the absence of exogenous copper, the requirement of the enzyme for ascorbate was more strict. In keeping with the proposed reaction mechanism, nearly 1 mol ascorbate was consumed for each mole of D-Tyr-Glu-NH2 produced.
authors.affiliation: Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: A S
authors.initials: AS
authors.lastname: Murthy
authors.forename: H T
authors.initials: HT
authors.lastname: Keutmann
authors.forename: B A
authors.initials: BA
authors.lastname: Eipper
mesh_terms: D000818:Animals; D002417:Cattle; D007202:Indicators and Reagents; D007700:Kinetics; D006899:Mixed Function Oxygenases; D009097:Multienzyme Complexes; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D007202:Indicators and Reagents; D009097:Multienzyme Complexes; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000587:Oxidoreductases Acting on CH-NH Group Donors