title: Correlation of copper valency with product formation in single turnovers of dopamine beta-monooxygenase.
abstract: Chemical- and freeze-quench EPR techniques have allowed single-turnover studies of the copper-containing enzyme dopamine beta-monooxygenase. Reduction of enzyme by a stoichiometric amount of ascorbate followed by rapid mixing with tyramine leads to oxidation of bound copper and formation of hydroxylated product in the expected 2:1 ratio. The tyramine dependence of single turnovers yields a limiting rate of 82 +/- 9 s-1 and Km of 3 +/- 1 mM, in agreement with kinetic modeling based on steady-state parameters. Together these results show that the reduced enzyme is a catalytically competent species, with bound copper acting as the sole reservoir of reducing equivalents. The correlation of copper oxidation and substrate hydroxylation rules out significant antiferromagnetic spin coupling in the enzyme-product complex. Since the enzyme-product complex's Cu2+ EPR signal is absent in the transient approach to the steady state [Brenner, M. C., Murray, C. J., & Klinman, J. P. (1989) Biochemistry (preceding paper in this issue)], this result implies that ascorbate reduces copper in the enzyme-product complex. These findings have important consequences for catalysis and active site structure in dopamine beta-monooxygenase.
authors.affiliation: Department of Chemistry, University of California, Berkeley 94720.
authors.forename: M C
authors.initials: MC
authors.lastname: Brenner
authors.forename: J P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D003300:Copper / Q000032:analysis / Q000378:metabolism*; D004299:Dopamine beta-Hydroxylase / Q000032:analysis / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005615:Freezing; D006900:Hydroxylation; D009655:Octopamine / Q000096:biosynthesis; D010084:Oxidation-Reduction; D014439:Tyramine / Q000378:metabolism
chemical_list: D009655:Octopamine; D003300:Copper; D004299:Dopamine beta-Hydroxylase; D001205:Ascorbic Acid; D014439:Tyramine