Kinetic Characterization of Prenyl-Flavin Synthase from &ITSaccharomyces cerevisiae&IT
Kinetic Characterization of Prenyl-Flavin Synthase from &ITSaccharomyces cerevisiae&IT
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DOI：
10.1021/acs.biochem.7b01131
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发表时间：
2018-02-06
期刊：
影响因子：
2.9
通讯作者：
Marsh, E. Neil G.
中科院分区：
文献类型：
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作者：
Arunrattanamook, Nattapol;Marsh, E. Neil G.
我们已经表征了来自酵母的异戊烯基黄素合酶的动力学和底物需求。这种酶催化将异戊烯基单元添加到还原的黄素单核苷酸（FMN）以形成桥接黄素核的NS和C6的额外的六元环，从而将黄素从氧化还原辅因子转化为支持芳基羧酸脱羧的辅因子。与细菌酶相反，酵母酶被发现使用二甲基烯丙基焦磷酸，而不是二甲基烯丙基磷酸，作为反应中的异戊二烯基供体。我们开发了一种联用测定异戊烯黄素合酶活性，其中营业额与异戊烯黄素依赖性酶阿魏酸脱羧酶的激活有关。该反应的动力学极其缓慢：k（cat）= 12.2 +/- 0.2 × 11 h（-1），二甲基烯丙基焦磷酸的K-M = 9.8 +/- 0.7 μ M。还原FMN的K-M测量得太准确。在预稳态条件下研究了减少FMN消耗的动力学。FMN的反应通过一级动力学很好地描述，k（app）为17.4 +/- 1.1 h（-1）。这些结果表明，一个化学步骤，最有可能形成的C6的黄素和异戊烯基部分之间的碳-碳键，是基本上在反应中的速率决定。
We have characterized the kinetics and substrate requirements of prenyl-flavin synthase from yeast. This enzyme catalyzes the addition of an isopentenyl unit to reduced flavin mononucleotide (FMN) to form an additional six-membered ring that bridges NS and C6 of the flavin nucleus, thereby converting the flavin from a redox cofactor to one that supports the decarboxylation of aryl carboxylic acids. In contrast to bacterial enzymes, the yeast enzyme was found to use dimethylallyl pyrophosphate, rather than dimethylallyl phosphate, as the prenyl donor in the reaction. We developed a coupled assay for prenylflavin synthase activity in which turnover was linked to the activation of the prenyl-flavin-dependent enzyme, ferulic acid decarboxylase. The kinetics of the reaction are extremely slow: k(cat) = 12.2 +/- 0.2 11 h(-1), and K-M for dimethylallyl pyrophosphate = 9.8 +/- 0.7 mu M. The K-M for reduced FMN was too accurately measured. The kinetics of reduced FMN consumption were studied under pre-steady state conditions. The reaction of FMN was described well by first-order kinetics with a k(app) of 17.4 +/- 1.1 h(-1). These results indicate that a chemical step, most likely formation of the carbon-carbon bond between C6 of the flavin and the isopentenyl moiety, is substantially rate-determining in the reaction.