Purification and characterization of avian dopamine beta-hydroxylase.
Dopamine beta-hydroxylase (EC 1.14.17.1) has been purified from the chromaffin granules of avian adrenals. The enzyme has a molecular mass of approximately 320K daltons and consists of four apparently identical subunits joined in pairs by disulfide bonds. Analysis of the products formed from dopamine tritiated in the beta position indicated that 1.72 times as much tritium was retained in norepinephrine as was released as water. Ferrocyanide could serve as a reductant, but ascorbate at equal concentrations afforded higher rates. The enzyme had a pH optimum of 5-6 and was activated by either fumarate or acetate, with fumarate being far more effective. Kinetic experiments varying the concentrations of the substrates ascorbate and dopamine and those of the products dehydroascorbate and norepinephrine suggested that the mechanism was un-uni bi-uni ping pong. By this mechanism, the enzyme released dehydroascorbate after being irreversibly reduced by ascorbate and then sequentially bound oxygen and dopamine and released the product norepinephrine. The enzyme was inhibited by high but probably physiological concentrations of the substrate ascorbate and was activated by low concentrations of the product dehydroascorbate. Ascorbate inhibition was noncompetitive with dopamine, and dehydroascorbate activation was due to an increase in the enzyme's affinity for ascorbate with little or no change in its Vmax. Substrate inhibition by ascorbate and product activation by dehydroascorbate might together ensure that the rate of norepinephrine synthesis in vivo remains relatively unaffected by changes in the ratio of ascorbate to dehydroascorbate within chromaffin granules.
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002645:Chickens; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D004299:Dopamine beta-Hydroxylase / Q000302:isolation & purification* / Q000378:metabolism; D008954:Models, Biological; D008970:Molecular Weight
D004299:Dopamine beta-Hydroxylase