pH dependence of peptidylglycine monooxygenase. Mechanistic implications of Cu-methionine binding dynamics

Andrew T. Bauman, Shula Jaron, Erik T. Yukl, Joel R. Burchfiel, Ninian J. Blackburn

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


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 pKa of 4.6, while C formed and decayed with pKas of 6.8 and 8.2, respectively. The pH dependence was found to be dominated by kCat, 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 pKas 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 pKa 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.

Original languageEnglish (US)
Pages (from-to)11140-11150
Number of pages11
Issue number37
StatePublished - Sep 19 2006

ASJC Scopus subject areas

  • Biochemistry


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