ESCHERICHIA-COLI PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM - EQUILIBRIUM KINETICS AND MECHANISM OF ENZYME-I PHOSPHORYLATION
ESCHERICHIA-COLI PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM - EQUILIBRIUM KINETICS AND MECHANISM OF ENZYME-I PHOSPHORYLATION
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DOI：
10.1021/bi00504a015
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发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
ROBILLARD, GT
中科院分区：
文献类型：
--
作者：
HOVING, H;LOLKEMA, JS;ROBILLARD, GT
酶I的磷酸化作用。通过磷酸烯醇式丙酮酸与丙酮酸之间的同位素交换研究了大肠杆菌磷酸烯醇式丙酮酸依赖性磷酸转移酶系统。监测1H-2 H交换的实验表明，酶I磷酸化伴随着质子从酶转移到底物的C-3原子。氘代和质子化丙酮酸的14 C-12 C交换实验显示出动力学同位素效应，表明质子转移是（部分）速率决定性的，并且是磷酰基转移机制中的重要步骤。在某些反应条件下，观察到14 C交换率随总酶浓度的增加而成比例增加，表明只有酶I的二聚体形式被磷酸化。根据14 C交换速率对磷酸烯醇丙酮酸和丙酮酸浓度的依赖性，确定反应的正向和反向二级速率常数为3 × 10 - 4。107和8 ×105 M-1 min-1，得到平衡常数apx。40和Δ G °。酶I磷酸化的[吉布斯自由能变化]为-2.3 kcal/mol。这些速率常数的值的磷酸转移酶系统的热力学的意义进行了讨论。
The phosphorylation of enzyme I from the E. coli phosphoenolpyruvate-dependent phosphotransferase system was studied by isotope exchange between phosphoenolpyruvate and pyruvate. Experiments monitoring 1H-2H exchange showed that enzyme I phosphorylation is accompanied by the transfer of a proton from the enzyme to the C-3 atom of the substrate. 14C-12C-exchange experiments with both deuterated and protonated pyruvate exhibited a kinetic isotope effect, showing that the proton transfer is (partly) rate determining and is an essential step in the mechanism of phosphoryl group transfer. Under certain reaction conditions, a more than proportional increase of the 14C exchange rate with increasing total enzyme concentration was observed, indicating that only the dimeric form of enzyme I is phosphorylated. From the dependence of the 14C exchange rate on the phosphoenolpyruvate and pyruvate concentrations, the forward and reverse 2nd-order rate constants of the reaction were determined to be 3 .times. 107 and 8 .times. 105 M-1 min-1, respectively, yielding an equilibrium constant of .apprx. 40 and a .DELTA.G.degree. [Gibbs free energy change] for enzyme I phosphorylation of -2.3 kcal/mol. The significance of the values of these rate constants for the thermodynamics of the phosphotransferase system is discussed.