Kinetics and mechanism of action of muscle pyruvate kinase.
Kinetics and mechanism of action of muscle pyruvate kinase.
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肌肉丙酮酸激酶的动力学和作用机制。
DOI：
10.1042/bj1690039
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发表时间：
1978
期刊：
影响因子：
--
通讯作者：
H. Britton
中科院分区：
文献类型：
--
作者：
L. Dann;H. Britton
1. 通过通量、同位素捕获、稳态速度和底物结合等方法研究了兔肌丙酮酸激酶的作用机制。所有测量均在Tris/HCl缓冲液pH8.5和5mm free Mg（2+）条件下进行。2. 介绍了以[(32)P]P(i)为原料高产备[(32)P]磷酸烯醇丙酮酸酯和测定[(32)P]-磷酸烯醇丙酮酸酯和[8-(14)C]ADP的方法。3. ATP与ADP的比值/ ATP与磷酸烯醇丙酮酸的比值（平衡状态下测量）随ADP浓度的增加呈双曲线增长，从1增加到2mm-ADP时的约2.1，但不受磷酸烯醇丙酮酸浓度的影响。由于比值大于1，添加底物的一个途径必须包括磷酸烯醇丙酮酸首先在限速步骤中添加到酶中。然而，底物也必须以替代顺序添加，因为ADP浓度的比例非线性增加，因为增加的速度远远小于从有序添加的稳态速度数据预测的速度。磷酸烯醇丙酮酸对比值的影响不大，这与替代途径中ADP的快速添加是一致的。在低ADP浓度下，替代途径对总反应的贡献小于33%。4. 用[(32)P]磷酸烯醇丙酮酸观察到同位素捕获，证实当磷酸烯醇丙酮酸首先加入酶时，它处于限速步骤。磷酸烯醇丙酮酸从三元配合物的释放也必须是一个缓慢的步骤。[8-(14)C]ADP未观察到捕获现象，因此除非ADP的解离常数非常大（约20mm），否则ADP加入游离酶的速度必须非常快。5. 结合研究表明，4mol的[(32)P]磷酸烯醇丙酮酸与1mol的酶结合，可能未与Mg（2+）结合，其解离常数与上述机制相符。未检测到[8-(14)C]ADP的结合，因此ADP的结合是通过低亲和力步骤进行的。后者也是稳态速度数据所要求的。6. 磷酸烯醇丙酮酸与ATP的通量/磷酸烯醇丙酮酸与丙酮酸的通量之比(由正向反应中[3-（14)C]-丙酮酸或[γ -(32)P]ATP的标签掺入磷酸烯醇丙酮酸测定）与单体没有显著差异。稳态速度数据预测了产品有序解离的通量比，结果表明解离必须是快速和随机的。数据还排除了乒乓机制。7. 计算了上述机制的允许速率常数。结果表明，无论底物的加入顺序如何，其结合的协同性都很高。
1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mm-free Mg(2+). 2. Methods of preparing [(32)P]phosphoenolpyruvate from [(32)P]P(i) in high yield and determining [(32)P]-phosphoenolpyruvate and [8-(14)C]ADP are described. 3. The ratio Flux of ATP to ADP/Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33% to the total reaction. 4. Isotope trapping was observed with [(32)P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-(14)C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mm). 5. Binding studies showed that 4mol of [(32)P]phosphoenolpyruvate binds to 1mol of the enzyme, probably unligated to Mg(2+), with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-(14)C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-(14)C]-pyruvate or [gamma-(32)P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.