Tittmann Kai, Wille Georg, Golbik Ralph, Weidner Annett, Ghisla Sandro, Hübner Gerhard
Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany.
Biochemistry. 2005 Oct 11;44(40):13291-303. doi: 10.1021/bi051058z.
The thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent pyruvate oxidase from Lactobacillus plantarum catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to acetyl-phosphate, carbon dioxide, and hydrogen peroxide. Central to the catalytic sequence, two reducing equivalents are transferred from the resonant carbanion/enamine forms of alpha-hydroxyethyl-ThDP to the adjacent flavin cofactor over a distance of approximately 7 A, followed by the phosphorolysis of the thereby formed acetyl-ThDP. Pre-steady-state and steady-state kinetics using time-resolved spectroscopy and a 1H NMR-based intermediate analysis indicate that both processes are kinetically coupled. In the presence of phosphate, intercofactor electron-transfer (ET) proceeds with an apparent first-order rate constant of 78 s(-1) and is kinetically gated by the preceding formation of the tetrahedral substrate-ThDP adduct 2-lactyl-ThDP and its decarboxylation. No transient flavin radicals are detectable in the reductive half-reaction. In contrast, when phosphate is absent, ET occurs in two discrete steps with apparent rate constants of 81 and 3 s(-1) and transient formation of a flavin semiquinone/hydroxyethyl-ThDP radical pair. Temperature dependence analysis according to the Marcus theory identifies the second step, the slow radical decay to be a true ET reaction. The redox potentials of the FAD(ox)/FAD(sq) (E1 = -37 mV) and FAD(sq)/FAD(red) (E2 = -87 mV) redox couples in the absence and presence of phosphate are identical. Both the Marcus analysis and fluorescence resonance energy-transfer studies using the fluorescent N3'-pyridyl-ThDP indicate the same cofactor distance in the presence or absence of phosphate. We deduce that the exclusive 10(2)-10(3)-fold rate enhancement of the second ET step is rather due to the nucleophilic attack of phosphate on the kinetically stabilized hydroxyethyl-ThDP radical resulting in a low-potential anion radical adduct than phosphate in a docking site being part of a through-bonded ET pathway in a stepwise mechanism of ET and phosphorolysis. Thus, LpPOX would constitute the first example of a radical-based phosphorolysis mechanism in biochemistry.
植物乳杆菌中依赖硫胺素二磷酸（ThDP）和黄素腺嘌呤二核苷酸（FAD）的丙酮酸氧化酶催化丙酮酸、无机磷酸和氧气转化为乙酰磷酸、二氧化碳和过氧化氢。在催化序列的核心部分，两个还原当量从α-羟乙基-ThDP的共振碳负离子/烯胺形式转移到相邻的黄素辅因子，转移距离约为7 Å，随后对由此形成的乙酰-ThDP进行磷酸解。使用时间分辨光谱和基于1H NMR的中间体分析进行的预稳态和稳态动力学表明，这两个过程在动力学上是耦合的。在磷酸盐存在的情况下，辅因子间电子转移（ET）以78 s(-1)的表观一级速率常数进行，并且在动力学上受四面体底物-ThDP加合物2-乳酰基-ThDP的先前形成及其脱羧作用的控制。在还原半反应中未检测到瞬态黄素自由基。相反，当不存在磷酸盐时，ET分两个离散步骤发生，表观速率常数分别为81和3 s(-1)，并瞬态形成黄素半醌/羟乙基-ThDP自由基对。根据马库斯理论进行的温度依赖性分析确定第二步，即缓慢的自由基衰减是真正的ET反应。在存在和不存在磷酸盐的情况下，FAD(ox)/FAD(sq)（E1 = -37 mV）和FAD(sq)/FAD(red)（E2 = -87 mV）氧化还原对的氧化还原电位相同。马库斯分析和使用荧光N3'-吡啶基-ThDP的荧光共振能量转移研究均表明，在存在或不存在磷酸盐的情况下，辅因子距离相同。我们推断，第二个ET步骤的10(2)-10(3)倍速率增强是由于磷酸盐对动力学稳定的羟乙基-ThDP自由基的亲核攻击导致形成低电位阴离子自由基加合物，而不是磷酸盐位于对接位点中作为逐步ET和磷酸解机制中通过键合ET途径的一部分。因此，LpPOX将构成生物化学中基于自由基的磷酸解机制的第一个例子。
