Negative Cooperativity in the Mechanism of Prenylated-Flavin-Dependent Ferulic Acid Decarboxylase: A Proposal for a “Two-Stroke” Decarboxylation Cycle
Negative Cooperativity in the Mechanism of Prenylated-Flavin-Dependent Ferulic Acid Decarboxylase: A Proposal for a “Two-Stroke” Decarboxylation Cycle
复制标题
异戊二烯化黄素依赖性阿魏酸脱羧酶机制中的负协同作用：“双冲程”脱羧循环的建议
DOI：
10.1021/acs.biochem.2c00460
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发表时间：
2023
期刊：
影响因子：
2.9
通讯作者：
Marsh, E. Neil
中科院分区：
文献类型：
--
作者：
Kaneshiro, April K.;Datar, Prathamesh M.;Marsh, E. Neil
阿魏酸脱羧酶（FDC）催化各种取代的苯基丙烯酸的可逆羧化反应，产生相应取代的苯乙烯和CO2。FDC是UbiD酶家族的成员，其使用异戊二烯化-FMN（prFMN）催化芳环和C-C双键上的脱羧反应。尽管越来越多的prFMN依赖性酶已被鉴定出来，但该反应的机制仍然知之甚少。在这里，我们提出了一个详细的预稳态动力学分析的FDC催化的反应prFMN与苯乙烯和苯基丙烯酸。基于所观察到的反应模式，我们提出了一个“两冲程”的动力学模型，其中FDC同源二聚体的两个亚基之间的负协同性在催化中起着重要的和以前未被认识到的作用。在该模型中，催化是在高亲和力的活性位点处引发的，其与苯基丙烯酸酯反应以在脱羧后产生共价结合的苯乙烯-prFMN环加合物。在催化循环的第二阶段中，第二底物分子与低亲和力活性位点的结合驱动构象转换，该构象转换使高亲和力和低亲和力活性位点相互转换。这种切换的亲和力夫妇的积极不利的环消除苯乙烯从第一个网站与积极有利的环加成和脱羧的苯基丙烯酸酯在第二个网站。我们注意到，作为一个警告，在这一点上，FDC动力学的复杂性留下开放的其他机制的解释，将需要进一步的实验，以更坚定地建立或反驳我们的建议。
Ferulic acid decarboxylase (FDC) catalyzes the reversible carboxylation of various substituted phenylacrylic acids to produce the correspondingly substituted styrenes and CO2. FDC is a member of the UbiD family of enzymes that use prenylated-FMN (prFMN) to catalyze decarboxylation reactions on aromatic rings and C–C double bonds. Although a growing number of prFMN-dependent enzymes have been identified, the mechanism of the reaction remains poorly understood. Here, we present a detailed pre-steady-state kinetic analysis of the FDC-catalyzed reaction of prFMN with both styrene and phenylacrylic acid. Based on the pattern of reactivity observed, we propose a “two-stroke” kinetic model in which negative cooperativity between the two subunits of the FDC homodimer plays an important and previously unrecognized role in catalysis. In this model, catalysis is initiated at the high-affinity active site, which reacts with phenylacrylate to yield, after decarboxylation, the covalently bound styrene–prFMN cycloadduct. In the second stage of the catalytic cycle, binding of the second substrate molecule to the low-affinity active site drives a conformational switch that interconverts the high-affinity and low-affinity active sites. This switching of affinity couples the energetically unfavorable cycloelimination of styrene from the first site with the energetically favorable cycloaddition and decarboxylation of phenylacrylate at the second site. We note as a caveat that, at this point, the complexity of the FDC kinetics leaves open other mechanistic interpretations and that further experiments will be needed to more firmly establish or refute our proposal.