Rapid freeze- and chemical-quench studies of dopamine beta-monooxygenase: comparison of pre-steady-state and steady-state parameters.
The copper-containing enzyme dopamine beta-monooxygenase has been studied with regard to pre-steady-state kinetics of tyramine hydroxylation and reduction of enzyme-bound Cu2+ by chemical- and freeze-quench EPR techniques. The bulk of the enzyme-bound copper (approximately 70%) is reduced in a single-exponential process with a limiting rate constant of 250 s-1, Km = 0.9 mM, consistent with participation of both copper ions in the redox events of catalysis. The remaining copper is reduced much more slowly (k approximately 2 s-1) or not at all, attributed to a distribution of copper into inhibitory binding sites and the presence of some inactive enzyme. Knowledge of the Cu2+ reduction rate, together with rate constants calculated from steady-state isotope effects [Miller, S. M., & Klinman, J. P. (1985) Biochemistry 24, 2114-2127], has allowed prediction of pre-steady-state product formation transients. Measurement of these transients under conditions of excess ascorbate shows close agreement with prediction, supporting the validity of individual rate constants obtained from steady-state data. Kinetic modeling shows further that the predominant steady-state enzyme form is the enzyme-product complex (E-P), which is expected to show a correspondingly large (approximately 70% of total copper) EPR signal for bound Cu2+. Surprisingly, the steady state is characterized by a low (19% of total copper) EPR signal. This lack of correlation between the anticipated and observed steady-state EPR signal suggests either antiferromagnetic coupling in binuclear copper centers or reduction of Cu2+ in this enzyme form by ascorbic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Brenner
M C
MC
Department of Chemistry, University of California, Berkeley 94720.
Murray
C J
CJ
Klinman
J P
JP
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001665:Binding Sites; D003198:Computer Simulation; D003300:Copper / Q000032:analysis*; D004299:Dopamine beta-Hydroxylase / Q000032:analysis*; D004578:Electron Spin Resonance Spectroscopy; D005615:Freezing; D006900:Hydroxylation; D007700:Kinetics; D010084:Oxidation-Reduction; D014439:Tyramine / Q000378:metabolism
D003300:Copper; D004299:Dopamine beta-Hydroxylase; D001205:Ascorbic Acid; D014439:Tyramine