pH Dependence of Peptidylglycine Monooxygenase. Mechanistic Implications of Cu−Methionine Binding Dynamics
| Autor: | Shula Jaron, Joel R. Burchfiel, Erik T. Yukl, Ninian J. Blackburn, Andrew T. Bauman |
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| Rok vydání: | 2006 |
| Předmět: |
Dansyl Compounds
chemistry.chemical_classification Binding Sites Methionine Absorption spectroscopy Stereochemistry Peptidylglycine monooxygenase Protonation Hydrogen-Ion Concentration Sulfonic acid Biochemistry Medicinal chemistry Acid dissociation constant Mixed Function Oxygenases chemistry.chemical_compound Deprotonation chemistry Multienzyme Complexes Enzyme kinetics Copper Protein Binding |
| Zdroj: | Biochemistry. 45:11140-11150 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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 pH9. 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. |
| Databáze: | OpenAIRE |
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