A pre-steady state kinetic analysis of the αY60W mutant of trans-3-chloroacrylic acid dehalogenase: implications for the mechanism of the wild-type enzyme.
A pre-steady state kinetic analysis of the αY60W mutant of trans-3-chloroacrylic acid dehalogenase: implications for the mechanism of the wild-type enzyme.
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DOI：
10.1021/bi3010686
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发表时间：
2012-11-20
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：
Huddleston JP;Schroeder GK;Johnson KA;Whitman CP
杀线虫剂1，3-二氯丙烯（一种异构体混合物）的细菌降解需要反式和顺式-3-氯丙烯酸脱卤酶（分别为CaaD和cis-CaaD）的作用。这两种酶都是互变异构酶超家族成员，并且共享3-卤代丙烯酸酯的相应异构体的水解脱卤的核心催化机制。顺式CaaD需要两个额外的残基的观察结果提出了一个问题，即CaaD如何用更少的催化残基进行类似的反应。作为确定明显更简单的CaaD催化反应的基础的努力的一部分，使用酶的荧光突变形式α Y 60 W-CaaD和反式-3-溴丙烯酸酯作为底物，通过停流和化学淬灭技术确定动力学机制。这些实验以及溴化物抑制研究的数据最好由六步模型提供，该模型提供了底物结合、化学和化学后发生的拟议构象变化的个体速率常数，然后释放丙二酸半醛和溴化物。构象变化和产物释放速率相当，它们共同限制了转换速率。动力学分析和建模研究验证了α Y 60 W-CaaD突变体作为酶催化反应过程中活性位点事件的准确报告者。α Y 60 W-CaaD催化反应的动力学机理与cis-CaaD催化反应的动力学机理相当。动力学模型和经验证的α Y 60 W-CaaD突变体为活性位点突变体的分析奠定了基础，以探索单个催化残基的贡献和简化反应的基础。
The bacterial degradation of the nematicide 1,3-dichloropropene, an isomeric mixture, requires the action of trans- and cis-3-chloracrylic acid dehalogenase (CaaD and cis-CaaD, respectively). Both enzymes are tautomerase superfamily members and share a core catalytic mechanism for the hydrolytic dehalogenation of the respective isomer of 3-haloacrylate. The observation that cis-CaaD requires two additional residues raises the question of how CaaD carries out a comparable reaction with fewer catalytic residues. As part of an effort to determine the basis for the apparently simpler CaaD-catalyzed reaction, the kinetic mechanism was determined by stopped-flow and chemical quench techniques using a fluorescent mutant form of the enzyme, αY60W-CaaD, and trans-3-bromoacrylate as the substrate. The data from these experiments as well as bromide inhibition studies are best accommodated by a six-step model that provides individual rate constants for substrate binding, chemistry, and a proposed conformational change occurring after chemistry followed by release of malonate semialdehyde and bromide. The conformational change and product release rates are comparable and together they limit the rate of turnover. The kinetic analysis and modeling studies validate the αY60W-CaaD mutant as an accurate reporter of active site events during the course of the enzyme-catalyzed reaction. The kinetic mechanism for the αY60W-CaaD-catalyzed reaction is comparable to that obtained for the cis-CaaD-catalyzed reaction. The kinetic model and the validated αY60W-CaaD mutant set the stage for an analysis of active site mutants to explore the contributions of individual catalytic residues and the basis for the simplicity of the reaction.
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影响因子：
2.9
作者：
Burks, Elizabeth A.;Fleming, Christopher D.;Mesecar, Andrew D.;Whitman, Christian P.;Pegan, Scott D.
通讯作者：
Pegan, Scott D.
DOI：
10.1099/00221287-137-8-2025
发表时间：
1991-08-01
期刊：
JOURNAL OF GENERAL MICROBIOLOGY
影响因子：
--
作者：
HARTMANS, S;JANSEN, MW;DEBONT, JAM
通讯作者：
DEBONT, JAM
影响因子：
2.9
作者：
Poelarends, Gerrit J.;Johnson, William H., Jr.;Whitman, Christian P.
通讯作者：
Whitman, Christian P.
影响因子：
2.9
作者：
Poelarends, GJ;Serrano, H;Whitman, CP
通讯作者：
Whitman, CP
影响因子：
2.9
作者：
Johnson, Kenneth A.;Simpson, Zachary B.;Blom, Thomas
通讯作者：
Blom, Thomas