Ash D E, Papadopoulos N J, Colombo G, Villafranca J J
J Biol Chem. 1984 Mar 25;259(6):3395-8.
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.
本研究探讨了与多巴胺β-羟化酶结合的铜的化学计量问题以及最大活性所需的铜原子数。将来自牛肾上腺髓质的四聚体酶与64Cu2+一起孵育，然后进行快速凝胶过滤，得到的酶每摩尔四聚体含有8.3 - 8.9摩尔铜。通过原子吸收法分析结合铜得到了相同的化学计量。利用核磁共振（NMR）和电子顺磁共振（EPR）监测用Cu2+对酶的滴定，结果表明溶剂水质子的纵向弛豫率和g约为2时信号的幅度在铜与蛋白质的比例达到约8之前呈线性增加。额外的滴定还表明，当每摩尔酶结合8摩尔Cu2+时，会形成一种酶 - Cu2+ - 酪胺 - CN - 抑制复合物。测量了基于机制的抑制剂2 - Br - 3 -（对羟基苯基）- 1 - 丙烯对多巴胺β-羟化酶的失活速率，并将其用作跟踪酶催化作用的一种方法。随着Cu2+增加，直至蛋白质与Cu2+的比例达到8 Cu/四聚体，观察到速率增加。达到该比例后，速率变得恒定。这些数据表明，多巴胺β-羟化酶特异性结合每四聚体8摩尔铜，并且这种化学计量是最大活性所必需的。
