Cromartie T H, Walsh C
Biochemistry. 1975 Jul 29;14(15):3482-9. doi: 10.1021/bi00686a030.
The falvoenzyme L-alpha-hydroxy acid oxidase from rat kidney [T.H Cromartie and C.T. Walsh (1975), Biochemistry 14, 2588] fails to catalyze the elimination of HCl form D,L-beta-chlorolactate, although this compound is a substrate for oxidation by the enzyme. Deuterium isotope effects demonstrate that proton removal from the alpha carbon of alpha-hydroxy acids is fully rate limiting, a finding in agreement with observations on L-lactate dehydrogenase from yeast [F. Lederer (1974), Eur. J. Biochem. 46, 393] which also does not promote elimination from D,L-beta-chlorolactate. Both D-alpha-hydroxy acid oxidase were found to be rapidly and irreversibly inactivated by the acetylenic substrate 1-hydroxy-3-butynoate. The partially purified dehydrogenase was observed to be inactivated within 10 min by 6.8 times 10(-8) M hydroxybutynoate. For the more extensively studied oxidase, inactivation was found to occur after 25 catalytic events, inactivation occurring by covalent addition of the inactivator to the coenzyme. A stoichimometry of one molecule of hydroxybutynoate per flavine was found, and the time course of inactivation was unaffected by the presence of thiols. The oxidase could also be inactivated by prolonged incubation of the enzyme with 2-hydroxy-3-butenoate, and inactivation which could be completely prevented by the presence of thiolds. Since the inactivation with hydroxybutenoate also left the flavine coenzyme unaltered, the inactivation was attributed to Michael addition of nucleophiles on the enzyme of the ketobutenoate product. Several 4-alkyl-substitued 2-hydroxy-3-butynoates were also observed to inactivate the oxidase by both coenzyme modification and random addition to the apoenzyme. It is proposed that the inactivation may occur by nucleophilic addition of a C4 allenic carbanion to the oxidized flavine coenzyme.
大鼠肾脏中的黄素酶L-α-羟基酸氧化酶[T.H. Cromartie和C.T. Walsh（1975年），《生物化学》14卷，2588页]不能催化从D,L-β-氯乳酸中消除HCl，尽管该化合物是该酶氧化的底物。氘同位素效应表明，从α-羟基酸的α-碳上去除质子是完全限速的，这一发现与对酵母L-乳酸脱氢酶的观察结果一致[F. Lederer（1974年），《欧洲生物化学杂志》46卷，393页]，该酶也不能促进从D,L-β-氯乳酸中消除。发现两种D-α-羟基酸氧化酶都被炔属底物1-羟基-3-丁炔酸迅速且不可逆地失活。观察到部分纯化的脱氢酶在10分钟内被6.8×10⁻⁸ M的羟基丁炔酸失活。对于研究更广泛的氧化酶，发现失活发生在25次催化事件之后，失活是通过失活剂与辅酶的共价加成而发生的。发现每分子黄素中有一分子羟基丁炔酸的化学计量关系，并且失活的时间进程不受硫醇存在的影响。该氧化酶也可通过将酶与2-羟基-3-丁烯酸长时间孵育而失活，并且硫醇的存在可完全防止这种失活。由于用羟基丁烯酸失活也使黄素辅酶保持不变，所以这种失活归因于酮丁烯酸产物在酶上的亲核加成。还观察到几种4-烷基取代的2-羟基-3-丁炔酸通过辅酶修饰和随机添加到脱辅基酶上而使氧化酶失活。有人提出，失活可能是通过C4丙二烯碳负离子对氧化的黄素辅酶的亲核加成而发生的。
