Stereospecific alkylation of cis-3-chloroacrylic acid dehalogenase by (R)-oxirane-2-carboxylate:: Analysis and mechanistic implications
Stereospecific alkylation of cis-3-chloroacrylic acid dehalogenase by (R)-oxirane-2-carboxylate:: Analysis and mechanistic implications
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DOI：
10.1021/bi049823h
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发表时间：
2004-06-08
期刊：
影响因子：
2.9
通讯作者：
Whitman, CP
中科院分区：
文献类型：
--
作者：
Poelarends, GJ;Serrano, H;Whitman, CP
反式 -3 - 氯丙烯酸脱卤酶（CaaD）和顺式 -3 - 氯丙烯酸脱卤酶（cis - CaaD）代表了细菌中两类主要的异构体选择性3 - 氯丙烯酸脱卤酶。它们催化反式或顺式 -3 - 卤代丙烯酸酯的水解脱卤反应，生成丙二酸半醛，推测是通过不稳定的卤代醇中间体进行的。鉴于针对这些酶提出的一种广义酸碱机制，（R） - 和（S） - 环氧乙烷 -2 - 羧酸酯被作为潜在的不可逆抑制剂进行了研究。只有顺式 - CaaD会以时间和浓度依赖的方式被不可逆抑制，并且仅被环氧乙烷 -2 - 羧酸酯的（R） - 对映体抑制。该酶呈现饱和动力学，并且底物的存在可使其免受失活。这些发现表明，失活过程涉及在活性位点首先形成一个可逆结合的酶 - 抑制剂复合物，随后发生共价修饰。对失活的顺式 - CaaD进行的质谱分析表明，Pro -1是修饰位点。还已确定Arg -70和Arg -73是共价修饰所必需的，因为R70A或R73A突变体与抑制剂一起孵育不会导致酶的烷基化。对野生型顺式 - CaaD动力学参数的pH依赖性研究表明，一个pKa约为9.3的质子化基团对催化至关重要。该基团可能是Pro -1，这使得它在失活实验条件下主要是一种带电物质。有两种机制可以解释这些观察结果。在一种机制中，环氧乙烷经历酸催化的开环，随后Pro -1的共轭碱被烷基化。或者，环氧乙烷发生亲核取代反应，其中Pro -1的共轭碱作为亲核试剂，酸催化剂使碳 - 氧键极化。这两个精氨酸残基可能结合羧酸酯基团，并使抑制剂处于有利于烷基化反应的取向。这些发现为对失活酶进行晶体学分析奠定了基础，以进一步阐明活性位点残基在失活过程和催化机制中的作用。
The enzymes trans-3-chloroacrylic acid dehalogenase (CaaD) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) represent the two major classes of bacterial, isomer-selective 3-chloroacrylic acid dehalogenases. They catalyze the hydrolytic dehalogenation of either trans- or cis-3-haloacrylates to yield malonate semialdehyde, presumably through unstable halohydrin intermediates. In view of a proposed general acid/base mechanism for these enzymes, (R)- and (S)-oxirane-2-carboxylate were investigated as potential irreversible inhibitors. Only cis-CaaD is irreversibly inhibited in a time- and concentration-dependent manner and only by the (R)-enantiomer of oxirane-2-carboxylate. The enzyme displays saturation kinetics and is protected from inactivation by the presence of substrate. These findings indicate that the inactivation process involves the initial formation of a reversibly bound enzyme- inhibitor complex at the active site followed by covalent modification. Mass spectral analysis of the inactivated cis-CaaD shows that Pro-1 is the site of modification. It has also been determined that Arg-70 and Arg-73 are required for covalent modification because incubation of either the R70A or R73A mutant with inhibitor does not result in enzyme alkylation. Studies of the pH dependence of the kinetic parameters of wild-type cis-CaaD reveal that a protonated group with a pK(a) of similar to9.3 is essential for catalysis. The group is likely Pro-1, making it predominately a charged species under the conditions of the inactivation experiments. Two mechanisms could account for these observations. In one mechanism, the oxirane undergoes acidcatalyzed ring opening followed by alkylation of the conjugate base of Pro-1. Alternatively, the oxirane undergoes a nucleophilic substitution reaction where the conjugate base of Pro-1 functions as the nucleophile and an acid catalyst polarizes the carbon oxygen bond. The two arginine residues likely bind the carboxylate group and position the inhibitor in a favorable orientation for the alkylation reaction. These findings set the stage for a crystallographic analysis of the inactived enzyme to delineate further the roles of active site residues in both the inactivation process and the catalytic mechanism.