Weiss P M, Garcia G A, Kenyon G L, Cleland W W, Cook P F
Department of Biochemistry, University of Wisconsin, Madison 53706.
Biochemistry. 1988 Mar 22;27(6):2197-205. doi: 10.1021/bi00406a058.
Benzoylformate decarboxylase (benzoylformate carboxy-lyase, BFD; EC 4.1.1.7) from Pseudomonas putida is a thiamine pyrophosphate (TPP) dependent enzyme which converts benzoylformate to benzaldehyde and carbon dioxide. The kinetics and mechanism of the benzoylformate decarboxylase reaction were studied by solvent deuterium and 13C kinetic isotope effects with benzoylformate and a series of substituted benzoylformates (pCH3O, pCH3, pCl, and mF). The reaction was found to have two partially rate-determining steps: initial tetrahedral adduct formation (D2O sensitive) and decarboxylation (13C sensitive). Solvent deuterium and 13C isotope effects indicate that electron-withdrawing substituents (pCl and mF) reduce the rate dependence upon decarboxylation such that decreased 13(V/K) effects are observed. Conversely, electron-donating substituents increase the rate dependence upon decarboxylation such that a larger 13(V/K) is seen while the D2O effects on V and V/K are not dramatically different from those for benzoylformate. All of the data are consistent with substituent stabilization or destabilization of the carbanionic intermediate (or carbanion-like transition state) formed during decarboxylation. Additional information regarding the mechanism of the enzymic reaction was obtained from pH studies on the reaction of benzoylformate and the binding of competitive inhibitors. These studies suggest that two enzymic bases are required to be in the correct protonation state (one protonated and one unprotonated) for optimal binding of substrate (or inhibitors).
恶臭假单胞菌的苯甲酰甲酸脱羧酶（苯甲酰甲酸羧基裂解酶，BFD；EC 4.1.1.7）是一种依赖硫胺焦磷酸（TPP）的酶，可将苯甲酰甲酸转化为苯甲醛和二氧化碳。通过对苯甲酰甲酸和一系列取代苯甲酰甲酸（对甲氧基苯、对甲苯、对氯苯和间氟苯）进行溶剂氘代和¹³C动力学同位素效应研究，探讨了苯甲酰甲酸脱羧酶反应的动力学和机制。发现该反应有两个部分速率决定步骤：初始四面体加合物形成（对D₂O敏感）和脱羧反应（对¹³C敏感）。溶剂氘代和¹³C同位素效应表明，吸电子取代基（对氯苯和间氟苯）降低了对脱羧反应的速率依赖性，从而观察到¹³(V/K)效应降低。相反，供电子取代基增加了对脱羧反应的速率依赖性，从而观察到更大的¹³(V/K)，而D₂O对V和V/K的影响与苯甲酰甲酸的影响没有显著差异。所有数据均与脱羧过程中形成的碳负离子中间体（或类碳负离子过渡态）的取代基稳定或不稳定作用一致。通过对苯甲酰甲酸反应和竞争性抑制剂结合进行pH研究，获得了有关酶促反应机制的更多信息。这些研究表明，两个酶碱基需要处于正确的质子化状态（一个质子化，一个未质子化）才能实现底物（或抑制剂）的最佳结合。
