title: pH Dependence of peptidylglycine monooxygenase. Mechanistic implications of Cu-methionine binding dynamics.
abstract: The pH dependence of the PHM-catalyzed monooxygenation of dansyl-YVG was studied in two different buffer systems in the pH range of 4-10. The pH-activity profile measured in a sulfonic acid buffer exhibited a maximum at pH 5.8 and became inactive at pH >9. The data could be fit to a model that assumed a protonated unreactive species A, a major reactive species B, and a less reactive species C. B formed in a deprotonation step with pK(a) of 4.6, while C formed and decayed with pK(a)s of 6.8 and 8.2, respectively. The pH dependence was found to be dominated by k(cat), with K(m)(dansyl-YVG) remaining pH-independent over the pH range of 5-8. Acetate-containing buffers shifted the pH maximum to 7.0, and the activity-pH profile could be simulated by formation and decay of a single active species with pK(a)s of 5.8 and 8.3, respectively. The pH-dependent changes in activity could be correlated with a change in the Debye-Waller factor for the Cu-S(met) (M314) component of the X-ray absorption spectrum which underwent a transition from a tightly bound inactive "met-on" form to a conformationally mobile active "met-off" form with a pK(a) which tracked the formation of the active species in both sulfonic acid and acetate-containing buffer systems. The data suggested that the conformational mobility of the bound substrate relative to the copper-superoxo active species is critical to catalysis and further suggested the presence of an accessible vibrational mode coupling Cu-S motion to the H tunneling probability along the Cu-O...H...C coordinate.
authors.affiliation: Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Sciences University, 20000 Northwest Walker Road, Beaverton, Oregon 97006-8921, USA.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Bauman
authors.forename: Shula
authors.initials: S
authors.lastname: Jaron
authors.forename: Erik T
authors.initials: ET
authors.lastname: Yukl
authors.forename: Joel R
authors.initials: JR
authors.lastname: Burchfiel
authors.forename: Ninian J
authors.initials: NJ
authors.lastname: Blackburn
mesh_terms: D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D003619:Dansyl Compounds / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D003619:Dansyl Compounds; D009097:Multienzyme Complexes; D003300:Copper; D008715:Methionine; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase