Division of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, Texas 78712, USA.
J Am Chem Soc. 2012 Jan 11;134(1):293-304. doi: 10.1021/ja206873f. Epub 2011 Dec 19.
cis-3-Chloroacrylic acid dehalogenase (cis-CaaD) catalyzes the hydrolytic dehalogenation of cis-3-haloacrylates to yield malonate semialdehyde. The enzyme processes other substrates including an allene (2,3-butadienoate) to produce acetoacetate. In the course of a stereochemical analysis of the cis-CaaD-catalyzed reaction using this allene, the enzyme was unexpectedly inactivated in the presence of NaBH(4) by the reduction of a covalent enzyme-substrate bond. Covalent modification was surprising because the accumulated evidence for cis-CaaD dehalogenation favored a mechanism involving direct substrate hydration mediated by Pro-1. However, the results of subsequent mechanistic, pre-steady state and full progress kinetic experiments are consistent with a mechanism in which an enamine forms between Pro-1 and the allene. Hydrolysis of the enamine or an imine tautomer produces acetoacetate. Reduction of the imine species is likely responsible for the observed enzyme inactivation. This is the first reported observation of a tautomerase superfamily member functioning by covalent catalysis. The results may suggest that some fraction of the cis-CaaD-catalyzed dehalogenation of cis-3-haloacrylates also proceeds by covalent catalysis.
顺式-3-氯丙烯酸脱卤酶（cis-CaaD）催化顺式-3-卤代丙烯酸盐的水解脱卤反应，生成丙二醛半醛。该酶还可以处理其他底物，包括丙二烯（2,3-丁二烯酸酯），以产生乙酰乙酸盐。在使用这种丙二烯对 cis-CaaD 催化反应进行立体化学分析的过程中，令人意外的是，在 NaBH4 的存在下，酶通过共价酶-底物键的还原而失活。共价修饰令人惊讶，因为 cis-CaaD 脱卤反应的累积证据支持一种涉及 Pro-1 介导的直接底物水合的机制。然而，随后的机制、预稳态和完整的动力学实验结果与一种机制一致，其中烯胺在 Pro-1 和丙二烯之间形成。烯胺或亚胺互变异构体的水解生成乙酰乙酸盐。亚胺物种的还原可能是观察到的酶失活的原因。这是首例报道的互变异构酶超家族成员通过共价催化作用的观察结果。结果可能表明，cis-CaaD 催化的 cis-3-卤代丙烯酸盐的脱卤反应的一部分也通过共价催化进行。
