STUDIES ON INTRAMOLECULAR AND INTERMOLECULAR KINETIC ISOTOPE EFFECTS IN PYRUVATE-CARBOXYLASE CATALYSIS
STUDIES ON INTRAMOLECULAR AND INTERMOLECULAR KINETIC ISOTOPE EFFECTS IN PYRUVATE-CARBOXYLASE CATALYSIS
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DOI：
10.1021/bi00662a017
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发表时间：
1976-01-01
期刊：
影响因子：
2.9
通讯作者：
WALSH, C
中科院分区：
文献类型：
--
作者：
CHEUNG, YF;WALSH, C
当以[2H3]丙酮酸为底物时，[鸡肝]丙酮酸羧化酶的2H动力学同位素效应为2.1。这种影响仅对Vmax/Km有影响，当衬底浓度为无穷大时，这种影响消失。在整个催化反应中，最慢的步骤显然是涉及酶-生物素被ATP和HCO3-羧化的半反应。~3H分子内同位素效应为4.8，分子间效应为1.2。前者被解释为有效催化常数[kcat]的同位素效应，而后者影响Vmax/Km。根据这些数据，估算了丙酮酸结合的RAE常数为4.5倍。106M~(-1)·min~(-1)，2H动力学同位素效应为3.1。得到了各速率常数的相对值。氟丙酮酸是一种反应速度慢6倍的底物。观察到2H动力学同位素效应为1.5，即使在无限底物浓度下也保持不变。整个催化反应中最慢的一步现在是氟丙酮酸的羧化。
A 2H kinetic isotope effect of 2.1 was observed when [2H3]pyruvate was used as the substrate for [chicken liver] pyruvate carboxylase. The effect is on Vmax/Km alone and disappears at infinite substrate concentration. The slowest step in the overall catalysis is apparently in the half-reaction involving the carboxylation of enzyme-biotin by ATP and HCO3-. A 3H intramolecular isotope effect of 4.8 and an intermolecular effect of 1.2 were also observed. The former was interpreted as the isotope effect on the effective catalytic constant [kcat], while the latter the one affected Vmax/Km. With these data, the rae constant for binding of pyruvate was estimated to be 4.5 .times. 106 M-1 min-1, and the 2H kinetic isotope effect on the catalytic step to be 3.1. Relative values for various rate constants were obtained. Fluoropyruvate was a substrate which reacted 6 times slower. A 2H kinetic isotope effect of 1.5 was observed, which remained even at infinite substrate concentration. The slowest step in the overall catalysis is now the carboxylation of fluoropyruvate.