Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine alpha-amidating enzyme.
Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the alpha-carbon of the glycyl residue, producing an alpha-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that alpha-AE requires 1.1 +/- 0. 2 mol of zinc/mol of enzyme for maximal (S)-N-dansyl-Tyr-Val-alpha-hydroxyglycine dealkylation activity. Treatment of the enzyme with EDTA abolishes both the peptide hydroxylation and the carbinolamide dealkylation activities. Addition of Zn(II), Co(II), Cd(II), and Mn(II) partially restores carbinolamide dealkylation activity to the EDTA-treated enzyme. Addition of Co(II) produces the greatest restoration of dealkylation activity, 32% relative to a control not treated with EDTA, while Mn(II) addition results in the smallest restoration of dealkylation activity, only 3% relative to an untreated control. The structure and coordination of the zinc center has been investigated by X-ray absorption spectroscopy. EXAFS data are best interpreted by an average coordination of 2-3 histidine ligands and 1-2 non-histidine O/N ligands. Since catalytic zinc centers in other zinc metalloenzymes generally exhibit only O/N ligands to the zinc atom, a zinc-bound water or hydroxide may serve as a general base for the abstraction of the hydroxyl proton from the carbinolamide intermediate. Alternatively, the zinc may function in a structural role.
Bell
J
J
Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania 15282, USA.
Ash
D E
DE
Snyder
L M
LM
Kulathila
R
R
Blackburn
N J
NJ
Merkler
D J
DJ
D000577:Amides / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000302:isolation & purification / Q000378:metabolism; D016466:CHO Cells; D003300:Copper / Q000378:metabolism; D006224:Cricetinae; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D051381:Rats; D013057:Spectrum Analysis; D014965:X-Rays; D015032:Zinc / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
D000577:Amides; D001059:Apoproteins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D003300:Copper; D004492:Edetic Acid; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D015032:Zinc