Pompon D, Lederer F
Eur J Biochem. 1985 Apr 1;148(1):145-54. doi: 10.1111/j.1432-1033.1985.tb08818.x.
The reaction of 2-hydroxy-3-butynoate, a suicide substrate, with flavocytochrome b2 [F. Lederer (1974) Eur. J. Biochem. 88, 393-399] has been reinvestigated. It is shown that no inactivation occurs under anaerobic conditions. In the presence of ferricyanide, the partition ratio between oxidation and inactivation is 3200. Ketobutynoate has no effect on oxidized flavocytochrome b2. But it inactivates the reduced enzyme, while undergoing catalytic reduction to hydroxybutynoate. The partition ratio between reduction and inactivation is 5. Inactivation followed by borohydride reduction was carried out in parallel with lactate oxidase and flavocytochrome b2. The decomposition products of the initial adduct formed between flavin and inactivator were isolated and characterized. One of them (compound II) was obtained from both enzymes and is identical to the one previously isolated from hydroxybutynoate-inactivated lactate oxidase [Schonbrunn et al. (1976) Biochemistry 15, 1798-1807]. Its decarboxylated derivative (compound I) was also formed. Another major adduct, compound III, was isolated only from flavocytochrome b2. Its structure and the conditions in which it appears suggest it is formed from the same primary adduct as compounds I and II, but by a different decomposition mode, on the enzyme itself. Altogether these results strengthen the idea that inactivation is caused by reaction between oxidized flavin and an allenic carbanion, the isomerisation product of a normal reaction intermediate. It is proposed that differences in the rate-determining step of the redox reaction explain the differences in the inactivation process which are observed between flavocytochrome b2, lactate oxidase and hydroxyacid oxidase.
对自杀底物2-羟基-3-丁炔酸酯与黄素细胞色素b2的反应[F. 莱德勒（1974年），《欧洲生物化学杂志》88卷，393 - 399页]进行了重新研究。结果表明，在厌氧条件下不会发生失活现象。在铁氰化物存在的情况下，氧化与失活之间的分配比为3200。酮丁炔酸酯对氧化型黄素细胞色素b2没有影响。但它会使还原型酶失活，同时自身催化还原为羟基丁炔酸酯。还原与失活之间的分配比为5。用硼氢化钠还原后失活的实验与乳酸氧化酶和黄素细胞色素b2同时进行。分离并鉴定了黄素与失活剂形成的初始加合物的分解产物。其中一种（化合物II）在两种酶中均有得到，且与先前从羟基丁炔酸酯失活的乳酸氧化酶中分离出的产物相同[舍恩布伦等人（1976年），《生物化学》15卷，1798 - 1807页]。其脱羧衍生物（化合物I）也已形成。另一种主要加合物，化合物III，仅从黄素细胞色素b2中分离得到。其结构以及出现的条件表明它与化合物I和II由相同的初级加合物形成，但在酶自身上通过不同的分解方式产生。总的来说，这些结果强化了这样一种观点，即失活是由氧化型黄素与烯丙基碳负离子（正常反应中间体的异构化产物）之间的反应引起的。有人提出，氧化还原反应中速率决定步骤的差异解释了在黄素细胞色素b2、乳酸氧化酶和羟基酸氧化酶之间观察到的失活过程的差异。
