title: Selective inactivation of the hydroxylase activity of bifunctional rat peptidylglycine alpha-amidating enzyme.
abstract: Conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 by recombinant type A rat 75-kDa peptidylglycine alpha-amidating enzyme (alpha-AE) is inactivated by ascorbate, dehydroascorbate, and hydrogen peroxide in a time- and concentration-dependent manner. Both ascorbate- and dehydroascorbate-mediated inactivation are saturable with apparent kinact/Kinact values of 1.7 and 0.23 s-1 M-1, respectively. Hydrogen peroxide-mediated inactivation is not saturable with a second-order rate constant of 50 s-1 M-1. Peptidyl-Gly substrates, EDTA, and H2O2 scavengers protect against ascorbate-mediated inactivation while EDTA and semidehydroascorbate scavengers protect against dehydroascorbate-mediated inactivation. Under similar conditions, ascorbate, dehydroascorbate, and H2O2 have no effect on the alpha-AE-catalyzed conversion of dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 which is consistent with the hypothesis that the 75-kDa enzyme consists of distinct peptidyl-Gly hydroxylase and peptidyl-alpha-hydroxyglycine lyase active sites.
authors.affiliation: Analytical Protein and Organic Chemistry Group, Unigene Laboratories, Inc., Fairfield, New Jersey 07004.
authors.forename: D J
authors.initials: DJ
authors.lastname: Merkler
authors.forename: R
authors.initials: R
authors.lastname: Kulathila
authors.forename: P P
authors.initials: PP
authors.lastname: Tamburini
authors.forename: S D
authors.initials: SD
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology; D002396:Catechols / Q000494:pharmacology*; D002460:Cell Line; D003683:Dehydroascorbic Acid / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007700:Kinetics; D051379:Mice; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009842:Oligopeptides; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000201:enzymology*; D014162:Transfection
chemical_list: D000975:Antioxidants; D002396:Catechols; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D006867:Hydrolases; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid