Klinman J P, Brenner M
Department of Chemistry, University of California, Berkeley 94720.
Prog Clin Biol Res. 1988;274:227-48.
Although classically characterized as a Type II copper protein, recent work has shown that D beta H requires two coppers per subunit for optimal activity. A major challenge has been to elucidate the relationship of these copper sites to one another, specifically whether there exists a single binuclear copper site or distinct metal sites catalyzing separate functions. In this paper, copper sites have been investigated by EPR techniques and rapid mixing methods to measure the rates and stoichiometries of copper reduction by ascorbate and subsequent reoxidation by tyramine. As shown, there is little or no evidence by EPR for spin coupling between metal sites in resting enzyme. Both coppers per subunit undergo reduction and reoxidation with single exponential values of 185 s-1 and 80 s-1, respectively. Significantly, the ratio of copper reoxidation to product formation in a single turnover is close to 2:1, implicating both coppers in the catalytic mechanism and ruling out any significant spin coupling in the enzyme-product complex. In contrast to single turnover experiments, rapid kinetics in the presence of excess ascorbate reveal a very low level of E-Cu (II). This result, which appears to conflict with evidence that the dominant enzyme form in the steady state is E-P, leads us to propose a reduction of enzyme at the level of E-P rather than free enzyme. Taken together with steady state kinetic parameters using alternate reductants, the data support separate binding sites for reductants and product/substrate and hence, separate functions for each copper per subunit. A detailed catalytic mechanism for D beta H is discussed in the context of a single metal site catalyzing dopamine hydroxylation.
尽管传统上被描述为一种II型铜蛋白，但最近的研究表明，多巴胺β羟化酶（D beta H）每个亚基需要两个铜离子才能达到最佳活性。一个主要挑战是阐明这些铜位点之间的相互关系，特别是是否存在一个单核双铜位点或催化不同功能的不同金属位点。在本文中，通过电子顺磁共振（EPR）技术和快速混合方法研究了铜位点，以测量抗坏血酸还原铜离子的速率和化学计量以及随后酪胺再氧化的情况。结果表明，EPR几乎没有证据表明静息酶中金属位点之间存在自旋耦合。每个亚基的两个铜离子都经历还原和再氧化，其单指数值分别为185 s-1和80 s-1。值得注意的是，在单次周转中铜再氧化与产物形成的比率接近2:1，这表明两个铜离子都参与催化机制，并排除了酶-产物复合物中任何显著的自旋耦合。与单次周转实验不同，在过量抗坏血酸存在下的快速动力学显示E-Cu（II）水平非常低。这一结果似乎与稳态下主要酶形式为E-P的证据相矛盾，这使我们提出在E-P水平而非游离酶水平上酶的还原。结合使用替代还原剂的稳态动力学参数，这些数据支持还原剂和产物/底物有单独的结合位点，因此每个亚基的每个铜离子具有不同的功能。本文在单个金属位点催化多巴胺羟化的背景下讨论了D beta H的详细催化机制。
