Cooperativity in the dopamine beta-monooxygenase reaction. Evidence for ascorbate regulation of enzyme activity.
The steady-state kinetic behavior of dopamine beta-monooxygenase (D beta M) has been examined over a 1000-fold range of ascorbate concentrations. Kinetic plots exhibit extreme curvature indicative of apparent negative cooperativity in the interaction of D beta M with ascorbate, with a calculated Hill coefficient of 0.15-0.30. The observed cooperativity is found to be independent of enzyme concentration and tyramine and oxygen concentrations, as well as the pH employed for the assay. Similar kinetic data have been obtained with both soluble and purified membrane-derived forms of enzyme. An investigation of the effect of the anion activator fumarate upon the observed kinetic patterns has demonstrated a conversion to a less cooperative kinetic pattern at low pH and high concentrations of fumarate. This phenomenon is attributed to an inhibitory binding of the structurally similar monoanionic species of fumarate to the ascorbate reductant site. A simple model has been used to assess the change in apparent Vmax and Km parameters with increased ascorbate concentrations. At all pH values examined, there is a dramatic decrease in the affinity of D beta M for ascorbate from a Km of approximately 0.05-0.10 mM (ascorbate concentration less than 1 mM) to Km greater than 10 mM at limiting ascorbate; at the same time there is a 3- to 4-fold increase in the limiting Vmax value. Several models have been considered to explain the observed activation of D beta M by high levels of ascorbic acid.
Stewart
L C
LC
Department of Chemistry, University of California, Berkeley 94720.
Klinman
J P
JP
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D001205:Ascorbic Acid / Q000502:physiology*; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D005650:Fumarates / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010100:Oxygen / Q000032:analysis
D005650:Fumarates; D004299:Dopamine beta-Hydroxylase; D001205:Ascorbic Acid; D010100:Oxygen