Department of Chemistry and Biochemistry, College of Pharmacy, University of Texas, Austin, Texas 78712, USA.
Biochemistry. 2009 Dec 15;48(49):11737-44. doi: 10.1021/bi901349z.
Isomer-specific 3-chloroacrylic acid dehalogenases catalyze the hydrolytic dehalogenation of the cis- and trans-isomers of 3-chloroacrylate to yield malonate semialdehyde. These reactions represent key steps in the degradation of the nematocide, 1,3-dichloropropene. The kinetic mechanism of cis-3-chloroacrylic acid dehalogenase (cis-CaaD) has now been examined using stopped-flow and chemical-quench techniques. Stopped-flow analysis of the reaction, following the fluorescence of an active site tryptophan, is consistent with a minimal three-step model involving substrate binding, chemistry, and product release. Chemical-quench experiments show burst kinetics, indicating that product release is at least partially rate limiting. Global fitting of all of the kinetic results by simulation is best accommodated by a four-step mechanism. In the final kinetic model, the enzyme binds substrate with an immediate isomerization to an alternate fluorescent form and chemistry occurs, followed by the ordered release of two products, with the release of the first product as the rate-limiting step. Bromide ion is a competitive inhibitor of the reaction indicating that it binds to the free enzyme rather than to the enzyme with one product still bound. This observation suggests that malonate semialdehyde is the first product released by the enzyme (rate limiting), followed by halide. A comparison of the unliganded cis-CaaD crystal structure with that of an inactivated cis-CaaD where the prolyl nitrogen of Pro-1 is covalently attached to (R)-2-hydroxypropanoate provides a possible explanation for the isomerization step. The structure of the covalently modified enzyme shows that a seven-residue loop comprised of residues 32-38 is closed down on the active site cavity where the backbone amides of two residues (Phe-37 and Leu-38) interact with the carboxylate group of the adduct. In the unliganded form, the same loop points away from the active site cavity. Similarly, substrate binding may cause this loop to close down on the active site and sequester the reaction from the external environment.
顺式-3-氯丙烯酸去卤酶能够特异性地催化顺式和反式-3-氯丙烯酸的水解脱卤反应，生成丙二醛半醛。这些反应是杀线虫剂 1,3-二氯丙烯降解的关键步骤。现在已经使用停流和化学猝灭技术研究了顺式-3-氯丙烯酸去卤酶（cis-CaaD）的动力学机制。通过荧光测定活性位点色氨酸的反应的停流分析与涉及底物结合、化学和产物释放的最小三步模型一致。化学猝灭实验显示爆发动力学，表明产物释放至少部分是速率限制步骤。通过模拟对所有动力学结果的全局拟合最好通过四步机制来解释。在最终的动力学模型中，酶与一个立即异构化的替代荧光形式结合，并进行化学转化，然后有序地释放两个产物，第一个产物的释放是限速步骤。溴化物离子是该反应的竞争性抑制剂，表明它与游离酶结合而不是与仍结合一个产物的酶结合。这一观察结果表明，丙二醛半醛是酶释放的第一个产物（限速），然后是卤化物。将未配位的顺式-CaaD 晶体结构与失活的顺式-CaaD 进行比较，其中 Pro-1 的脯氨酸氮共价连接到（R）-2-羟基丙酸酯，这为异构化步骤提供了一个可能的解释。共价修饰酶的结构表明，由残基 32-38 组成的包含七个残基的环关闭在活性位点腔上，其中两个残基（Phe-37 和 Leu-38）的酰胺基与加合物的羧基相互作用。在未配位的形式中，相同的环指向远离活性位点腔。同样，底物结合可能导致该环在活性位点上关闭，并将反应与外部环境隔离。
