Investigation of the carboligase activity of thiamine diphosphate-dependent enzymes using kinetic modeling and NMR spectroscopy
Investigation of the carboligase activity of thiamine diphosphate-dependent enzymes using kinetic modeling and NMR spectroscopy
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DOI：
10.1016/j.molcatb.2009.02.021
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发表时间：
2009-11-01
影响因子：
--
通讯作者：
Pohl, Martina
中科院分区：
文献类型：
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作者：
Kokova, Mariya;Zavrel, Michael;Pohl, Martina
通过初始速率测定、反应过程曲线分析和基于NMR的反应中间体分析研究了二磷酸硫胺素（ThDP）依赖性酶苯甲醛裂解酶（BAL）和苯甲酰甲酸脱羧酶变体His281Ala（BFDH281A）催化的安息香缩合反应。使用的机械动力学模型，动力学参数和微观速率常数进行了测定，从而确定反应的速率限制步骤。在BAL中，整个反应受产物释放的速率限制，而在BFDH281A中，底物结合是最慢的催化步骤。这些结果通过在酸淬灭分离后使用NMR光谱分析共价反应中间体来进一步证实。(C)2009 Elsevier B.V.保留所有权利。
The benzoin condensation reaction catalyzed by the thiamine diphosphate (ThDP)-dependent enzymes benzaldehyde lyase (BAL) and benzoylformate decarboxylase variant His281Ala (BFDH281A) was studied via initial rate measurements, progress curve analysis and NMR-based analysis of reaction intermediates. Using a mechanistic kinetic model, the kinetic parameters and microscopic rate constants were determined, thus identifying the rate limiting steps of the reaction. In BAL, overall reaction is rate-limited by product release, whereas in BFDH281A Substrate binding is the slowest step of catalysis. These results were further confirmed by analysis of covalent reaction intermediates Using NMR spectroscopy after acid quench isolation. (C) 2009 Elsevier B.V. All rights reserved.