Kinetic Analysis of Transient Intermediates in the Mechanism of Prenyl-Flavin-Dependent Ferulic Acid Decarboxylase
Kinetic Analysis of Transient Intermediates in the Mechanism of Prenyl-Flavin-Dependent Ferulic Acid Decarboxylase
复制标题
DOI：
10.1021/acs.biochem.0c00856
复制
发表时间：
2021-01-19
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：
--
作者：
Kaneshiro, April K.;Koebke, Karl J.;Marsh, E. Neil G.
阿魏酸脱羧酶使用最近发现的辅因子异戊二烯化的FMN（prFMN）催化各种取代的苯基丙烯酸脱羧为它们相应的苯乙烯衍生物和CO2。该机制涉及prFMN和底物之间不寻常的1，3-偶极环加成反应，以产生能够进行脱羧的环加成物。使用本机质谱，我们表明酶形成一个稳定的prFMN-苯乙烯环加合物，积累在营业额的酶。预稳态动力学分析的反应，使用紫外可见光停流光谱揭示了一个复杂的模式的动力学行为，最好的描述了一个半的网站模型，涉及负的二聚体酶的两个亚基之间的协同性。对于反应性位点，prFMN与苯丙烯酸的环加合物以131 s（-1）的k（app）形成。该中间体转化为prFMN-苯乙烯环加合物，k（app）为75 s（-1）。生成苯乙烯和游离酶的prFMN-苯乙烯环加合物的环消除似乎决定了总反应的k（cat），其为11.3 s（-1）。
Ferulic acid decarboxylase catalyzes the decarboxylation of various substituted phenylacrylic acids to their corresponding styrene derivatives and CO2 using the recently discovered cofactor prenylated FMN (prFMN). The mechanism involves an unusual 1,3-dipolar cycloaddition reaction between prFMN and the substrate to generate a cycloadduct capable of undergoing decarboxylation. Using native mass spectrometry, we show the enzyme forms a stable prFMN-styrene cycloadduct that accumulates on the enzyme during turnover. Pre-steady state kinetic analysis of the reaction using ultraviolet-visible stopped-flow spectroscopy reveals a complex pattern of kinetic behavior, best described by a half-of-sites model involving negative cooperativity between the two subunits of the dimeric enzyme. For the reactive site, the cycloadduct of prFMN with phenylacylic acid is formed with a k(app) of 131 s(-1). This intermediate converts to the prFMN-styrene cycloadduct with a k(app) of 75 s(-1). Cycloelimination of the prFMN-styrene cycloadduct to generate styrene and free enzyme appears to determine k(cat) for the overall reaction, which is 11.3 s(-1).