Bifunctional peptidylglcine alpha-amidating enzyme requires two copper atoms for maximum activity.
The conversion of C-terminal glycine-extended peptides to C-terminal alpha-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 residue and the second step is the dealkylation of the alpha-hydroxyglycine-extended peptide to the alpha-amidated peptide and glyoxylate. We show 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 (approximately 80 microM) required to measure initial rates for this poor substrate. The enzyme, as purified, contains a substoichiometric amount of copper and has only trace levels of amidation activity. Addition of exogenous Cu(II) ions stimulates amidation activity approximately 3000-fold at the optimum copper stoichiometry and the enzyme is then inhibited by excess Cu(II). No stimulation of amidation activity is observed upon the addition of the following divalent metal ions: Mn(II), Fe(II), Ni(II), Cd(II), and the oxovanadium cation, VO(II). The enzyme-catalyzed dealkylation of alpha-hydroxyhippuric acid to benzamide shows no dependence on copper, indicating that the copper dependence of the amidation reaction must be attributed to a copper dependence in peptide alpha-hydroxylation.
Kulathila
R
R
Analytical Protein and Organic Chemistry Group, Unigene Laboratories, Inc., Fairfield, New Jersey 07004.
Consalvo
A P
AP
Fitzpatrick
P F
PF
Freeman
J C
JC
Snyder
L M
LM
Villafranca
J J
JJ
Merkler
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002413:Cations, Divalent; D003300:Copper / Q000494:pharmacology*; D006224:Cricetinae; D003619:Dansyl Compounds / Q000378:metabolism; D005456:Fluorescent Dyes; D005998:Glycine / Q000378:metabolism; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009842:Oligopeptides / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000201:enzymology
D002413:Cations, Divalent; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011994:Recombinant Proteins; D003300:Copper; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D005998:Glycine