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

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

Research output: Contribution to journalArticle

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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 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
JournalBiochemistry
Volume45
Issue number37
DOIs
StatePublished - Sep 19 2006

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Methionine
Buffers
Sulfonic Acids
Acetates
Deprotonation
X ray absorption
Electron transitions
Catalysis
Copper
Absorption spectra
peptidylglycine monooxygenase
Substrates
X-Rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

pH dependence of peptidylglycine monooxygenase. Mechanistic implications of Cu-methionine binding dynamics. / Bauman, Andrew T.; Jaron, Shula; Yukl, Erik T.; Burchfiel, Joel R.; Blackburn, Ninian.

In: Biochemistry, Vol. 45, No. 37, 19.09.2006, p. 11140-11150.

Research output: Contribution to journalArticle

Bauman, Andrew T. ; Jaron, Shula ; Yukl, Erik T. ; Burchfiel, Joel R. ; Blackburn, Ninian. / pH dependence of peptidylglycine monooxygenase. Mechanistic implications of Cu-methionine binding dynamics. In: Biochemistry. 2006 ; Vol. 45, No. 37. pp. 11140-11150.
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AB - 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.

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