Daff S, Sharp R E, Short D M, Bell C, White P, Manson F D, Reid G A, Chapman S K
Department of Chemistry, University of Edinburgh, Scotland, UK.
Biochemistry. 1996 May 21;35(20):6351-7. doi: 10.1021/bi9522561.
Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. At 25 degrees C, 0.10 M ionic strength, and saturating L-lactate concentration, the turnover rate is 207 s-1 [per cytochrome c reduced; Miles, C. S., Rouviere, N., Lederer, F., Mathews, F. S., Reid, G. A., Black, M. T., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. The second-order rate constant for cytochrome c reduction in the pre-steady-state has been determined by stopped-flow spectrophotometry to be 34.8 (+/- 0.9) muM-1 s-1 in the presence of 10 mM L-lactate. This rate constant has been found to be dependent entirely on the rate of complex formation, the electron-transfer rate in the pre-formed complex being in excess of 1000 s-1. Inhibition of the pre-steady-state reduction of cytochrome c by either zinc-substituted cytochrome c or ferrocytochrome c has led to the estimation of a Kd for the catalytically competent complex of 8 microM, and from this the dissociation rate constant of 280 s-1, a value much less than the actual electron-transfer rate. The inhibition observed is only partial which indicates that electron transfer from the 1:1 complex to another cytochrome c can occur and that alternative electron transfer sites exist. The cytochrome c binding site proposed by Tegoni et al. [Tegoni, M., White, S. A., Roussel, A., Mathews, F. S. & Cambillau, C. (1993) Proteins 16, 408-422] has been tested using site-directed mutagenesis. Mutations designed to affect the complex stability and putative electron-transfer pathway had little effect, suggesting that the primary cytochrome c binding site on flavocytochrome b2 lies elsewhere. The combination of tight binding and multiple electron-transfer sites gives flavocytochrome b2 a low K(m) and a high kcat, maximizing its catalytic efficiency. In the steady-state, the turnover rate is therefore largely limited by other steps in the catalytic cycle, a conclusion which is discussed in the preceding paper in this issue [Daff, S., Ingledew, W. J., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6345-6350].
来自酿酒酵母的黄素细胞色素b2将L-乳酸脱氢与细胞色素c还原偶联起来。在25摄氏度、离子强度为0.10 M以及L-乳酸浓度饱和的条件下，周转速率为207 s-1[每还原一个细胞色素c；迈尔斯，C.S.，鲁维耶尔，N.，勒德雷尔，F.，马修斯，F.S.，里德，G.A.，布莱克，M.T.，&查普曼，S.K.（1992年）《生物化学杂志》285，187 - 192]。在预稳态下细胞色素c还原的二级速率常数已通过停流分光光度法测定，在存在10 mM L-乳酸的情况下为34.8（±0.9）μM-1 s-1。已发现该速率常数完全取决于复合物形成的速率，预形成复合物中的电子转移速率超过1000 s-1。锌取代的细胞色素c或亚铁细胞色素c对细胞色素c预稳态还原的抑制作用已导致对催化活性复合物的Kd估计为8 μM，并由此得出解离速率常数为280 s-1，该值远低于实际电子转移速率。观察到的抑制只是部分抑制，这表明从1:1复合物到另一个细胞色素c的电子转移可以发生，并且存在替代的电子转移位点。特戈尼等人[特戈尼，M.，怀特，S.A.，鲁塞尔，A.，马修斯，F.S. &坎比劳，C.（1993年）《蛋白质》16，408 - 422]提出的细胞色素c结合位点已通过定点诱变进行了测试。旨在影响复合物稳定性和假定电子转移途径的突变几乎没有影响，这表明黄素细胞色素b2上的主要细胞色素c结合位点位于其他地方。紧密结合和多个电子转移位点的组合赋予黄素细胞色素b2低Km和高kcat，使其催化效率最大化。因此，在稳态下，周转速率在很大程度上受到催化循环中其他步骤的限制，这一结论在本期的前一篇论文中进行了讨论[达夫，S.，英格利德，W.J.，里德，G.A.，&查普曼，S.K.（1996年）《生物化学》35，6345 - 6350]。
