Kinetic and spectroscopic studies of the interaction of copper with dopamine beta-hydroxylase.
The question of the stoichiometry of copper bound to dopamine beta-hydroxylase and the number of copper atoms required for maximal activity was addressed in this study. Incubation of tetrameric enzyme from bovine adrenal medulla with 64Cu2+ followed by rapid gel filtration yielded an enzyme containing 8.3-8.9 mol of Cu/mol of tetramer. An identical stoichiometry was obtained by analysis of bound copper by atomic absorption methods. NMR and EPR were used to monitor titrations of the enzyme with Cu2+ and showed that the longitudinal relaxation rate of solvent water protons and the amplitude of the signal at g approximately 2 increased linearly up to a copper to protein ratio of approximately 8. Additional titrations also indicate that an enzyme-Cu2+-tyramine-CN- inhibitory complex was formed when 8 mol of Cu2+ are bound per mol of enzyme. The rate of inactivation of dopamine beta-hydroxylase by the mechanism-based inhibitor 2-Br-3-(p-hydroxyphenyl)-1-propene was measured and used as a method to follow enzymatic catalysis. An increase in rate was observed with increasing Cu2+ up to a protein to Cu2+ ratio of 8 Cu/tetramer. The rate becomes constant after this ratio is achieved. These data indicate that dopamine beta-hydroxylase specifically binds 8 mol of Cu/tetramer and that this stoichiometry is required for maximal activity.
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology; D003300:Copper / Q000378:metabolism* / Q000494:pharmacology; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D046911:Macromolecular Substances; D010636:Phenols / Q000494:pharmacology; D011485:Protein Binding
C040825:2-bromo-(4-hydroxyphenyl)-1-propene; D046911:Macromolecular Substances; D010636:Phenols; D003300:Copper; D004299:Dopamine beta-Hydroxylase; C029892:cupric chloride