来自兔肌肉的2,3-二磷酸甘油酸依赖性磷酸甘油酸变位酶的作用机制。

Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle.

作者信息

Britton H G, Clarke J B

出版信息

Biochem J. 1972 Nov;130(2):397-410. doi: 10.1042/bj1300397.

DOI:10.1042/bj1300397

PMID:4677138

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1174419/

Abstract

The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of (32)P- and (14)C-labelled substrates at chemical equilibrium. 3. With (14)C-labelled substrates no induced transport was found over a wide concentration range, and with (32)P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. (14)C-labelled substrates exchange at twice the rate of (32)P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4x10(6)s(-1). The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the K(m) for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase.

摘要

讨论了2,3 - 二磷酸甘油酸依赖性磷酸甘油酸变位酶的性质和动力学。该反应至少有三种可能的机制：（i）磷酸酶（乒乓）机制；（ii）磷酸从2,3 - 二磷酸甘油酸向底物的分子间转移（顺序机制）；（iii）磷酸的分子内转移。得出结论，这些机制无法通过传统的动力学测量来区分。2. 计算了不同机制的通量，结果表明，通过适当的诱导转运试验以及比较化学平衡时(32)P和(14)C标记底物的通量，应该能够区分这些机制。3. 使用(14)C标记底物时，在很宽的浓度范围内未发现诱导转运，而使用(32)P标记底物时，发生了共转运，其在两倍浓度范围内与浓度无关。在化学平衡时，(14)C标记底物的交换速率是(32)P标记底物的两倍。结果完全符合磷酸酶机制，并表明磷酸酶异构化的速率常数不小于4×10(6)s(-1)。完全排除了磷酸的分子内转移（以及底物的两个或更多分子之间的分子间转移）。只有当2 - 磷酸甘油酸的K(m)比观察值小7.5倍以上，并且酶 - 2,3 - 二磷酸甘油酸复合物的异构化是反应中的主要限速步骤时，磷酸从2,3 - 二磷酸甘油酸的分子间转移才与结果相符。4. 实验证明的磷酸酶非常快速的异构化表明了一种不涉及形式异构化的机制。根据这个新方案，该酶在机制上可能在进化上与2,3 - 二磷酸甘油酸二磷酸酶密切相关。