Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine α-amidating enzyme

Joseph Bell, David E. Ash, Lynn M. Snyder, Raviraj Kulathila, Ninian Blackburn, David J. Merkler

Research output: Contribution to journalArticle

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Abstract

Bifunctional peptidylglycine α-amidating enzyme (α-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal α- amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the α-carbon of the glycyl residue, producing an α-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that α-AE requires 1.1 ± 0.2 mol of zinc/mol of enzyme for maximal (S)-N-dansyl-Tyr-Val-α-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.

Original languageEnglish (US)
Pages (from-to)16239-16246
Number of pages8
JournalBiochemistry
Volume36
Issue number51
DOIs
StatePublished - 1997

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Dealkylation
Zinc
Rats
Enzymes
Edetic Acid
Hydroxylation
Peptides
Ligands
Histidine
Restoration
Copper
X-Ray Absorption Spectroscopy
X ray absorption spectroscopy
Hydroxyl Radical
Glycine
Protons
Carbon
Atoms
Water

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine α-amidating enzyme. / Bell, Joseph; Ash, David E.; Snyder, Lynn M.; Kulathila, Raviraj; Blackburn, Ninian; Merkler, David J.

In: Biochemistry, Vol. 36, No. 51, 1997, p. 16239-16246.

Research output: Contribution to journalArticle

Bell, Joseph ; Ash, David E. ; Snyder, Lynn M. ; Kulathila, Raviraj ; Blackburn, Ninian ; Merkler, David J. / Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine α-amidating enzyme. In: Biochemistry. 1997 ; Vol. 36, No. 51. pp. 16239-16246.
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T1 - Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine α-amidating enzyme

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AU - Merkler, David J.

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N2 - Bifunctional peptidylglycine α-amidating enzyme (α-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal α- amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the α-carbon of the glycyl residue, producing an α-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that α-AE requires 1.1 ± 0.2 mol of zinc/mol of enzyme for maximal (S)-N-dansyl-Tyr-Val-α-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.

AB - Bifunctional peptidylglycine α-amidating enzyme (α-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal α- amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the α-carbon of the glycyl residue, producing an α-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that α-AE requires 1.1 ± 0.2 mol of zinc/mol of enzyme for maximal (S)-N-dansyl-Tyr-Val-α-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.

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