KINETIC AND MAGNETIC RESONANCE STUDIES OF THE PYRUVATE KINASE REACTION. I. DIVALENT METAL COMPLEXES OF PYRUVATE KINASE.
KINETIC AND MAGNETIC RESONANCE STUDIES OF THE PYRUVATE KINASE REACTION. I. DIVALENT METAL COMPLEXES OF PYRUVATE KINASE.
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丙酮酸激酶反应的动力学和磁共振研究。
DOI：
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发表时间：
1965
影响因子：
4.8
通讯作者：
M. Cohn
中科院分区：
文献类型：
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作者：
A. Mildvan;M. Cohn
丙酮酸激酶反应机制的许多方面已得到阐明（l），但之前的研究并未集中在反应所需的二价（2）和一价（3）阳离子的作用上。关于二价阳离子，尚未确定酶是否作为催化金属-底物络合物反应的“游离”酶起作用，或反之亦然。根据两个动力学观察结果：（a）镁超过核苷酸时不会抑制丙酮酸激酶反应，（5）镁对ADP的Ki有很小的影响（4，5），Prudard、Hass、Jacobsen和Boyer（4）提出腺嘌呤核苷酸可以作为其镁盐参与反应。后一项发现表明，相反的情况可能成立;即，ADP不会显着影响镁的K。这种行为是由二价阳离子和核苷酸的酶的独立结合引起的（6）。因此，很明显，仅动力学研究通常不足以提供在两种可能的活性物质（金属酶或金属底物）之间做出决定的证据（7）。目前的工作进行，以阐明二价阳离子在丙酮酸激酶反应中的作用。除了镁，其他二价阳离子激活丙酮酸激酶，产生较低的观察到的速度。因此，Solvonuk和Collier（8）比较了5 mM水平的二价阳离子，发现了以下速度顺序。镁大于锰，锰大于钴;锌没有活性。钙已被证明是丙酮酸激酶的有效抑制剂（9）。在这项研究中选择顺磁性锰离子作为活化剂，因为该离子的络合物可以通过磁共振方法（lo-12）进行研究，从而可以比较通过这些平衡方法获得的解离常数与动力学常数。本系列的第一篇论文是对酶和Mn 2 * 之间的二元复合物的研究。随后的论文将描述酶，锰和底物之间的三元复合物的性质。
Many aspects of the mechanism of the pyruvate kinase reaction have been elucidated (l), but previous investigations have not been focused on the roles of the divalent (2) and monovalent (3) cations required for the reaction. With respect to the divalent cation, it has not been ascertained whether the enzyme functions as a “free” enzyme catalyzing the reaction of a metal-substrate complex or vice versa. Reynard, Hass, Jacobsen, and Boyer (4) have suggested that adenine nucleotides can participate in the reaction as their magnesium salts on the basis of two kinetic observations: (a) magnesium in excess of nucleotides does not inhibit the pyruvate kinase reaction and (5) magnesium has a small effect on the K;, of ADP (4, 5). The latter finding suggests that the converse probably holds; namely, that ADP does not significantly affect the K, of magnesium. Such behavior would result from independent binding by the enzyme of the divalent cation and the nucleotide (6). It is clear, therefore, that kinetic studies alone generally provide insufficient evidence to decide between the two possible active species, metal enzyme or metal substrate (7). The present work was undertaken to elucidate the role of the divalent cation in the pyruvate kinase reaction. In addition to magnesium, other divalent cations activate pyruvate kinase, producing lower observed velocit.ies. Thus, Solvonuk and Collier (8), comparing 5 mM levels of the divalent cations, found the following order of velocities. Magnesium was greater than manganese which was greater than cobalt; zinc showed no activity. Calcium has been shown to be a potent inhibitor of pyruvate kinase (9). The paramagnetic manganous ion was chosen as activator in this study because the complexes of this ion may be studied by magnetic resonance methods (lo-12), permitting a comparison of the dissociation constants obtained by these equilibrium methods with kinetic constants. The first paper of this series is a study of the binary complex between the enzyme and Mn2*. A subsequent paper will describe the properties of the ternary complexes between the enzyme, manganese, and substrates.