Alliel P M, Mulet C, Lederer F
Eur J Biochem. 1980 Apr;105(2):343-51. doi: 10.1111/j.1432-1033.1980.tb04507.x.
We have reported in a previous communication a kinetic study showing bromopyruvate to behave as an active-site-directed reagent for flavocytochrome b2. It is shown here that inactivation is accompanied by incorporation of 3 mol reagent/subunit of oxidized intact enzyme and 4 mol reagent/subunit nicked enzyme. Only one of the modifications is presumed to be responsible for activity loss. All labeled groups are found to be cysteines. Incubation of reduced nicked enzyme with bromopyruvate results in total protection of activity and loss of only one sulfhydryl group. A subsequent incubation in the presence of the competitive inhibitor sulfite leads to some more loss of non-essential groups. After these two pretreatments, incubation in the presence of bromo[2-14C]pyruvate results in incorporation of 1.2--1.5 mol reagent/subunit concomitant with the loss of about 0.8 active site. A study of the distribution of label between fragments alpha and beta has been carried out using gel electrophoresis and Sephadex filtration after selective proteolysis. It is shown that the active-site sulfhydryl group corresponds to one of the four cysteines situated in the last two thirds of fragment alpha. The structural and functional implications of these results is discussed.
我们在之前的一篇通讯中报道了一项动力学研究，该研究表明溴丙酮酸可作为黄素细胞色素b2的活性位点导向试剂。本文表明，失活伴随着每亚基完整氧化酶掺入3摩尔试剂以及每亚基切口酶掺入4摩尔试剂。据推测，只有一种修饰导致活性丧失。所有标记基团均为半胱氨酸。用溴丙酮酸孵育还原的切口酶可完全保护活性，且仅损失一个巯基。随后在竞争性抑制剂亚硫酸盐存在下孵育会导致一些非必需基团进一步损失。经过这两种预处理后，在溴[2-14C]丙酮酸存在下孵育会导致每亚基掺入1.2 - 1.5摩尔试剂，同时约0.8个活性位点丧失。在选择性蛋白水解后，使用凝胶电泳和葡聚糖凝胶过滤对α和β片段之间的标记分布进行了研究。结果表明，活性位点巯基对应于位于α片段最后三分之二处的四个半胱氨酸之一。讨论了这些结果的结构和功能意义。
