The catalytic core of peptidylglycine alpha-hydroxylating monooxygenase: investigation by site-directed mutagenesis, Cu X-ray absorption spectroscopy, and electron paramagnetic resonance.
Peptidylglycine alpha-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 beta-monooxygenase (D beta M) was characterized. Mutation of Tyr79, the residue equivalent to a p-cresol target in D beta M, to Phe79 altered the kinetic parameters of PHM. Disruption of either His-rich cluster contained within the PHM/D beta M 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 D beta M. Mutation of the Met residues common to PHM and D beta M to Ile identified Met314 as critical for catalytic activity.
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Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Quon
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Mains
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Boswell
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Blackburn
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002417:Cattle; D002460:Cell Line; D003300:Copper / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry; D051381:Rats; D013057:Spectrum Analysis; D013379:Substrate Specificity; D014965:X-Rays
D009097:Multienzyme Complexes; D009842:Oligopeptides; D003300:Copper; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase