Ferguson Kyle L, Arunrattanamook Nattapol, Marsh E Neil G
Department of Chemistry, ‡Department of Chemical Engineering, and §Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
Biochemistry. 2016 May 24;55(20):2857-63. doi: 10.1021/acs.biochem.6b00170. Epub 2016 May 10.
Ferulic acid decarboxylase from Saccharomyces cerevisiae catalyzes the decarboxylation of phenylacrylic acid to form styrene using a newly described prenylated flavin mononucleotide cofactor. A mechanism has been proposed, involving an unprecedented 1,3-dipolar cyclo-addition of the prenylated flavin with the α═β bond of the substrate that serves to activate the substrate toward decarboxylation. We measured a combination of secondary deuterium kinetic isotope effects (KIEs) at the α- and β-positions of phenylacrylic acid together with solvent deuterium KIEs. The solvent KIE is 3.3 on Vmax/KM but is close to unity on Vmax, indicating that proton transfer to the product occurs before the rate-determining step. The secondary KIEs are normal at both the α- and β-positions but vary in magnitude depending on whether the reaction is performed in H2O or D2O. In D2O, the enzyme catalyzed the exchange of deuterium into styrene; this reaction was dependent on the presence of bicarbonate. This observation implies that CO2 release must occur after protonation of the product. Further information was obtained from a linear free-energy analysis of the reaction through the use of a range of para- and meta-substituted phenylacrylic acids. Log(kcat/KM) for the reaction correlated well with the Hammett σ(-) parameter with ρ = -0.39 ± 0.03; r(2) = 0.93. The negative ρ value and secondary isotope effects are consistent with the rate-determining step being the formation of styrene from the prenylated flavin-product adduct through a cyclo-elimination reaction.
来自酿酒酵母的阿魏酸脱羧酶利用一种新描述的异戊烯基化黄素单核苷酸辅因子催化苯丙烯酸脱羧形成苯乙烯。有人提出了一种机制，涉及异戊烯基化黄素与底物的α═β键进行前所未有的1,3-偶极环加成，从而使底物易于脱羧。我们测量了苯丙烯酸α位和β位的二级氘动力学同位素效应（KIEs）以及溶剂氘KIEs的组合。溶剂KIE在Vmax/KM上为3.3，但在Vmax上接近1，这表明质子转移到产物发生在速率决定步骤之前。α位和β位的二级KIEs均为正常，但大小因反应是在H2O还是D2O中进行而有所不同。在D2O中，该酶催化氘与苯乙烯的交换；此反应依赖于碳酸氢盐的存在。这一观察结果意味着CO2的释放必须在产物质子化之后发生。通过使用一系列对位和间位取代的苯丙烯酸对该反应进行线性自由能分析，获得了更多信息。该反应的Log(kcat/KM)与哈米特σ(-)参数相关性良好，ρ = -0.39 ± 0.03；r(2) = 0.93。负的ρ值和二级同位素效应与速率决定步骤是通过环消除反应从异戊烯基化黄素 - 产物加合物形成苯乙烯一致。
