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首页> 外文期刊>Biochemistry >PHOSPHOENOLPYRUVATE MUTASE CATALYSIS OF PHOSPHORYL TRANSFER IN PHOSPHOENOLPYRUVATE - KINETICS AND MECHANISM OF PHOSPHORUS-CARBON BOND FORMATION
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PHOSPHOENOLPYRUVATE MUTASE CATALYSIS OF PHOSPHORYL TRANSFER IN PHOSPHOENOLPYRUVATE - KINETICS AND MECHANISM OF PHOSPHORUS-CARBON BOND FORMATION

机译：磷磷酸丙酮酸酯的磷酸磷酸突变酶催化-磷-碳键形成的动力学和机理。

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Phosphoenolpyruvate phosphomutase (PEP mutase) from Tetrahymena pyriformis catalyzes the rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr). A spectrophotometric P-pyr assay consisting of the coupled actions of P-pyr decarboxylase, phosphonoacetaldehyde hydrolase, and alcohol dehydrogenase was devised to monitor mutase catalysis. The reaction constants determined for PEP mutase catalyzed conversion of PEP to P-pyr at pH 7.5 and 25 degrees C in the presence of MS(II) are k(cat) = 5 s(-1), K-m = 0.77 +/- 0.05 mM, and K-eq = (2-9) x 10(-4) In the PEP forming direction, k(cat) = 100 (-1) and K-m = 3.5 +/- 0.1 mu M. Retention of stereochemistry at phosphorus and strong inhibition displayed s by the pyruvyl enolate analog, oxalate, have been cited as two lines of evidence that PEP mutase catalysis proceeds via a phosphoenzyme-pyruvyl enolate intermediate [Seidel, H. M., & Knowles, J. R. (1994) Biochemistry 33, 5641-5646]. In this study, single turnover reactions of oxalyl phosphate with the PEP mutase were carried out to test the formation of the phosphoenzyme intermediate. If formed, the phosphoenzyme-oxalate complex should be sufficiently stable to isolate. Reaction of the mutase with [P-32]oxalyl phosphate in the presence of Mg(II)/Mn(II) cofactor failed to produce a detectable level of the [P-32]phosphoenzyme-oxalate complex. In contrast, the same reaction carried out with pyruvate phosphate dikinase (PPDK), an enzyme known to catalyze the phosphorylation of its active site histidine with PEP, occurred at a rate of 4 x 10(-4) s(-1) (15% E-P formed) in the presence Mg(II) and at a rate of 3 x 10(-3) s(-1) (60% E-P formed) in the presence of Mn(II). Both oxalyl phosphate (K-i = 180 +/- 10 mu M) and oxalate (K-i = 32 +/- 10 mu M) were competitive inhibitors of PEP mutase catalysis, but neither displayed slow, tight binding inhibition. These results do not support the intermediacy of a phosphoenzyme-pyruvyl enolate complex in PEP mutase catalysis.
机译：梨形四膜虫中的磷酸烯醇丙酮酸磷酸突变酶（PEP mutase）催化磷酸烯醇丙酮酸（PEP）重排成磷酸壬丙酮酸（P-pyr）。设计了由P-pyr脱羧酶，膦酰基乙醛水解酶和醇脱氢酶的耦合作用组成的分光光度法P-pyr分析，以监测突变酶的催化作用。在MS（II）存在下，在pH 7.5和25摄氏度下，PEP突变酶催化PEP转化为P-pyr的反应常数为k（cat）= 5 s（-1），Km = 0.77 +/- 0.05 mM，且K-eq =（2-9）x 10（-4）在PEP形成方向上，k（cat）= 100（-1），Km = 3.5 +/- 0.1μM。丙酮酸烯醇酯类似物草酸盐所显示的强烈抑制作用已被引用为两条证据，表明PEP突变酶催化作用是通过磷酸酶-丙酮酸烯醇酯中间体进行的[Seidel，HM，＆Knowles，JR（1994）Biochemistry 33，5641- 5646]。在这项研究中，草酸磷酸酯与PEP突变酶进行了单周转反应，以测试磷酸酶中间体的形成。如果形成，则磷酸酶-草酸盐复合物应足够稳定以分离。在Mg（II）/ Mn（II）辅因子存在下，突变酶与[P-32]草酰磷酸的反应未能产生可检测水平的[P-32]磷酸酶-草酸酯复合物。相反，用丙酮酸磷酸二激酶（PPDK）进行的同一反应以4 x 10（-4）s（-1）的速率发生，该酶已知可催化其活性位点组氨酸被PEP磷酸化（15）。在存在Mg（II）的情况下，生成3％的EP），在Mn（II）存在的情况下，以3 x 10（-3）s（-1）的速率生成60％的EP。草酸磷酸酯（K-i = 180 +/- 10μM）和草酸酯（K-i = 32 +/- 10μM）都是PEP突变酶催化的竞争性抑制剂，但均未显示出缓慢，紧密的结合抑制作用。这些结果不支持在PEP突变酶催化中磷酸酶-丙酮酸烯醇酯复合物的中介作用。

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