The Catalytic Core of Peptidylglycine α-Hydroxylating Monooxygenase: Investigation by Site-Directed Mutagenesis, Cu X-ray Absorption Spectroscopy, and Electron Paramagnetic Resonance

Betty A. Eipper, Andrew S.W. Quon, Richard E. Mains, John S. Boswell, Ninian J. Blackburn

Research output: Contribution to journalArticle

94 Scopus citations

Abstract

Peptidylglycine α-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that plays a key role in the biosynthesis of many peptides. Using site-directed mutagenesis, the catalytic core of PHM was found not to extend beyond Asp359. Shorter PHM proteins were eliminated intracellularly, suggesting that they failed to fold correctly. A set of mutant PHM proteins whose design was based on the structural and mechanistic similarities of PHM and dopamine β-monooxygenase (DβM) was characterized. Mutation of Tyr79, the residue equivalent to a p-cresol target in DβM, to Phe79 altered the kinetic parameters of PHM. Disruption of either His-rich cluster contained within the PHM/DβM homology domain eliminated activity, while deletion of a third His-rich cluster unique to PHM failed to affect activity; the catalytically inactive mutant PHM proteins still bound to a peptidylglycine substrate affinity resin. EPR and EXAFS studies of oxidized PHM indicate that the active site contains type 2 copper in a tetragonal environment; the copper is coordinated to two to three His and one to two additional O/N ligands, probably solvent, again supporting the structural homology of PHM and DβM. Mutation of the Met residues common to PHM and DβM to He identified Met314 as critical for catalytic activity.

Original languageEnglish (US)
Pages (from-to)2857-2865
Number of pages9
JournalBiochemistry
Volume34
Issue number9
DOIs
StatePublished - Mar 1995

ASJC Scopus subject areas

  • Biochemistry

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