Structural and Functional Investigations on the Role of Zinc in Bifunctional Rat Peptidylglycine -Amidating Enzyme

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• 作者：Joseph Bell ; David E. Ash ; Lynn M. Snyder ; Raviraj Kulathila ; Ninian J. Blackburn ; and David J. Merkler
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：December 23, 1997
• 年：1997
• 卷：36
• 期：51
• 页码：16239 - 16246
• 全文大小：161K
• 年卷期：v.36,no.51(December 23, 1997)
• ISSN：1520-4995

文摘

Bifunctional peptidylglycine -amidating enzyme(-AE) catalyzes the two-step conversionof C-terminal glycine-extended peptides to C-terminal -amidatedpeptides and glyoxylate. The firststep is the ascorbate-, O₂-, and copper-dependenthydroxylation of the -carbon of the glycyl residue,producing an -hydroxyglycine-extended peptide. The second stepis the ascorbate-, O₂-, and copper-independent dealkylation of the carbinolamide intermediate. We showthat -AE requires 1.1 ± 0.2 molof zinc/mol of enzyme for maximal(S)-N-dansyl-Tyr-Val--hydroxyglycinedealkylation activity.Treatment of the enzyme with EDTA abolishes both the peptidehydroxylation and the carbinolamidedealkylation activities. Addition of Zn(II), Co(II),Cd(II), and Mn(II) partially restorescarbinolamidedealkylation activity to the EDTA-treated enzyme. Addition ofCo(II) produces the greatest restorationof dealkylation activity, 32% relative to a control not treated withEDTA, while Mn(II) addition resultsin the smallest restoration of dealkylation activity, only 3% relativeto an untreated control. The structureand coordination of the zinc center has been investigated by X-rayabsorption spectroscopy. EXAFSdata are best interpreted by an average coordination of 2-3 histidineligands and 1-2 non-histidine O/Nligands. Since catalytic zinc centers in other zinc metalloenzymesgenerally exhibit only O/N ligands tothe zinc atom, a zinc-bound water or hydroxide may serve as a generalbase for the abstraction of thehydroxyl proton from the carbinolamide intermediate.Alternatively, the zinc may function in a structuralrole.