title: Mechanistic investigation of peptidylglycine alpha-hydroxylating monooxygenase via intrinsic tryptophan fluorescence and mutagenesis.
abstract: The biosynthesis of the majority of biologically active peptides ends with an obligatory alpha-amidation step that is catalyzed only by peptidylglycine alpha-hydroxylating monooxygenase (PHM). The utility of two mechanisms proposed for this copper- and ascorbate-dependent monooxygenase was examined using site-directed mutagenesis and intrinsic tryptophan fluorescence. Retention of full activity by PHMccGln(170)Ala and -Asn eliminates a critical role for Gln(170) in a substrate-mediated electron transfer pathway. The 20-fold reduction in V(max) observed for PHMccGln(170)Glu and -Leu is consistent with a key role for conformational changes in this region. Mutation of Tyr(79), situated near Cu(A), to Trp reduced V(max) 200-fold. Measurement of changes in intrinsic fluorescence allowed determination of a K(d) for copper (0.06 microM) and for a peptidylglycine substrate, Phe-Gly-Phe-Gly (0.8 microM). Although the peptidylglycine substrate bound more tightly at pH 7.0 than at pH 5.5, V(max) decreased 25-fold at neutral pH. Total quenching of the signal from Trp(79) in apoPHMccTyr(79)Trp along with its greatly reduced V(max) defines a critical role for Cu(A) in the rate-limiting step of the reaction. Taking into account our data and the results of kinetic, spectroscopic, and crystallographic studies, we propose a mechanism in which substrate-mediated activation of molecular oxygen binding at Cu(A) completes a pathway for electron transfer from Cu(B).
authors.affiliation: Department of Neuroscience, The University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Bell
authors.forename: Rajaâ
authors.initials: R
authors.lastname: El Meskini
authors.forename: Darlene
authors.initials: D
authors.lastname: D'Amato
authors.forename: Richard E
authors.initials: RE
authors.lastname: Mains
authors.forename: Betty A
authors.initials: BA
authors.lastname: Eipper
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002384:Catalysis; D002460:Cell Line; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D009097:Multienzyme Complexes; D009842:Oligopeptides; D011994:Recombinant Proteins; D003300:Copper; D014364:Tryptophan; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D005998:Glycine