Phenylpyruvate tautomerase activity of trans-3-chloroacrylic acid dehalogenase:: Evidence for an enol intermediate in the dehalogenase reaction?
Phenylpyruvate tautomerase activity of trans-3-chloroacrylic acid dehalogenase:: Evidence for an enol intermediate in the dehalogenase reaction?
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DOI：
10.1021/bi7007189
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发表时间：
2007-08-21
期刊：
影响因子：
2.9
通讯作者：
Whitman, Christian P.
中科院分区：
文献类型：
--
作者：
Poelarends, Gerrit J.;Johnson, William H., Jr.;Whitman, Christian P.
顺式 - 或反式 - 3 - 氯丙烯酸向丙二酸半醛的酶促转化是杀线虫剂1,3 - 二氯丙烯细菌降解的关键步骤。对于负责这一步骤的异构体特异性水解脱卤酶，即顺式 - 和反式 - 3 - 氯丙烯酸脱卤酶（分别为cis - CaaD和CaaD），已经提出了两种机制。在一种机制中，丙二酸半醛的烯醇异构体是通过从最初的卤代醇物质中α,β - 消除HCl产生的。现在发现苯烯醇丙酮酸是CaaD的底物，其k(cat)/K - m值接近使用反式 - 3 - 氯丙烯酸酯测定的CaaD反应的值。此外，该反应具有立体选择性，在（H₂O） - H - 2中以1.8 : 1的比例生成[3 - H - 2]苯丙酮酸的3S异构体。这两个观察结果以及对CaaD活性位点突变体的动力学分析表明，CaaD的活性位点负责苯丙酮酸互变异构酶（PPT）活性。这种活性是催化混杂性的一个显著例子，并且可能反映了在CaaD介导的反式 - 3 - 氯丙烯酸酯脱卤反应中存在烯醇中间体。CaaD和顺式 - CaaD在互变异构酶超家族中代表不同的家族，这是一组结构同源的蛋白质，其特征是具有核心β - α - β结构单元和一个催化性的Pro - 1。被称为巨噬细胞迁移抑制因子（MIF）的真核免疫调节蛋白也是互变异构酶超家族成员，表现出PPT活性，但生物学相关性未知。除了机制上的意义外，这些结果在CaaD和超家族互变异构酶之间建立了功能联系，突出了β - α - β支架的催化和结合混杂性，并表明MIF的PPT活性可能反映了在一个未知的MIF催化反应中的部分反应。
The enzymatic conversion of cis- or trans-3-chloroacrylic acid to malonate semialdehyde is a key step in the bacterial degradation of the nematocide 1,3-dichloropropene. Two mechanisms have been proposed for the isomer-specific hydrolytic dehalogenases, cis- and trans-3-chloroacrylic acid dehalogenase (cis-CaaD and CaaD, respectively), responsible for this step. In one mechanism, the enol isomer of malonate semialdehyde is produced by the alpha,beta-elimination of HCl from an initial halohydrin species. Phenylenolpyruvate has now been found to be a substrate for CaaD with a k(cat)/K-m value that approaches the one determined for the CaaD reaction using trans-3-chloroacrylate. Moreover, the reaction is stereoselective, generating the 3S isomer of [3-H-2]phenylpyruvate in a 1.8:1 ratio in (H2O)-H-2. These two observations and a kinetic analysis of active site mutants of CaaD suggest that the active site of CaaD is responsible for the phenylpyruvate tautomerase (PPT) activity. The activity is a striking example of catalytic promiscuity and could reflect the presence of an enol intermediate in CaaD-mediated dehalogenation of trans-3-chloroacrylate. CaaD and cis-CaaD represent different families in the tautomerase superfamily, a group of structurally homologous proteins characterized by a core beta-alpha-beta building block and a catalytic Pro-1. The eukaryotic immunoregulatory protein known as macrophage migration inhibitory factor (MIF), also a tautomerase superfamily member, exhibits a PPT activity, but the biological relevance is unknown. In addition to the mechanistic implications, these results establish a functional link between CaaD and the superfamily tautomerases, highlight the catalytic and binding promiscuity of the beta-alpha-beta scaffold, and suggest that the PPT activity of MIF could reflect a partial reaction in an unknown MIF-catalyzed reaction.