18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine alpha-amidating enzyme is a monooxygenase.
The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.
Merkler
D J
DJ
Analytical Protein & Organic Chemistry Group, Unigene Laboratories, Inc., Fairfield, New Jersey 07004.
Kulathila
R
R
Consalvo
A P
AP
Young
S D
SD
Ash
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002247:Carbon Isotopes; D002460:Cell Line; D003619:Dansyl Compounds / Q000378:metabolism; D006038:Glyoxylates / Q000378:metabolism; D007700:Kinetics; D007773:Lactates / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009492:Neurospora crassa / Q000201:enzymology; D009842:Oligopeptides / Q000378:metabolism; D010103:Oxygen Isotopes; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013964:Thyroid Neoplasms / Q000201:enzymology; D014162:Transfection
D002247:Carbon Isotopes; D003619:Dansyl Compounds; D006038:Glyoxylates; D007773:Lactates; D009097:Multienzyme Complexes; D009842:Oligopeptides; D010103:Oxygen Isotopes; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D001205:Ascorbic Acid