Fitzpatrick P F, Harpel M R, Villafranca J J
Arch Biochem Biophys. 1986 Aug 15;249(1):70-5. doi: 10.1016/0003-9861(86)90561-8.
In order to determine the order of substrate binding to dopamine beta-hydroxylase during catalysis, the effect of alternate substrates upon kinetic parameters was examined. The V/K value for ascorbate was unchanged when tyramine, phenylpropylamine, p-Cl-phenethylamine, p-CH3O-phenethylamine, or phenethylamine was the hydroxylated substrate. The V/K values for tyramine and oxygen were similarly unchanged when ferrocyanide was used as the reductant in place of ascorbate. In order to use ferrocyanide as reductant it was necessary to include copper to alleviate the substrate inhibition seen with this substrate. The pattern of substrate inhibition observed with ferrocyanide was consistent with a small amount of free cyanide present in the ferrocyanide. With ferrocyanide as reductant and [2,2-2H2]tyramine as substrate, there was a measurable isotope effect on the V/K value for oxygen, but none on the values of Vmax or V/K for tyramine. These results are consistent with a ping-pong mechanism in which tyramine binds to the enzyme after the release of oxidized ascorbate. Subsequently, oxygen binds to form a ternary complex.
为了确定催化过程中底物与多巴胺β-羟化酶结合的顺序，研究了替代底物对动力学参数的影响。当酪胺、苯丙胺、对氯苯乙胺、对甲氧基苯乙胺或苯乙胺为羟基化底物时，抗坏血酸的V/K值不变。当用亚铁氰化物代替抗坏血酸作为还原剂时，酪胺和氧气的V/K值同样不变。为了使用亚铁氰化物作为还原剂，必须加入铜以减轻该底物引起的底物抑制。观察到的亚铁氰化物引起的底物抑制模式与亚铁氰化物中存在少量游离氰化物一致。以亚铁氰化物作为还原剂，[2,2-2H2]酪胺作为底物，对氧气的V/K值有可测量的同位素效应，但对酪胺的Vmax或V/K值没有影响。这些结果与乒乓机制一致，即酪胺在氧化型抗坏血酸释放后与酶结合。随后，氧气结合形成三元复合物。
