Monitoring the timing of ATP hydrolysis with activation of peptide cleavage in Escherichia coli Lon by transient kinetics.
Monitoring the timing of ATP hydrolysis with activation of peptide cleavage in Escherichia coli Lon by transient kinetics.
复制标题
通过瞬态动力学监测大肠杆菌 Lon 中肽裂解激活的 ATP 水解时间。
DOI：
10.1021/bi048618z
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Lee,Irene
中科院分区：
文献类型：
--
作者：
Vineyard,Diana;Patterson-Ward,Jessica;Berdis,AnthonyJ;Lee,Irene
大肠杆菌，也称为蛋白酶 La，是一种寡聚 ATP 依赖性蛋白酶，其功能是降解细胞中受损的和某些短命的调节蛋白。研究E的动力学机制。大肠杆菌蛋白酶，我们对该酶的 ATP 酶和肽酶活性进行了首次稳态前动力学表征。使用快速猝灭流和荧光停流光谱技术，我们证明了 ATP 水解发生在肽裂解之前，前一个反应显示出爆发，而后者显示出产物生产的滞后。 ATP 水解中突发动力学的检测表明核苷酸水解后的步骤是 ATP 酶周转的限速。在饱和底物浓度下，肽裂解的滞后速率常数与 ATP 酶的 kcat 相当，表明在第一次酶周转期间两个水解过程是协调的。酶催化过程中亚基相互作用的参与被检测为底物结合和水解的正协同性，以及 Lon 中 ATP 酶活性的明显不对称性。当我们的数据放在一起时，它们与反应模型一致，其中 ATP 水解用于生成水解肽的活性酶形式。
Escherichia coliLon, also known as protease La, is an oligomeric ATP-dependent protease, which functions to degrade damaged and certain short-lived regulatory proteins in the cell. To investigate the kinetic mechanism ofE. coliLon protease, we performed the first pre-steady-state kinetic characterization of the ATPase and peptidase activities of this enzyme. Using rapid quench-flow and fluorescence stopped-flow spectroscopy techniques, we demonstrated that ATP hydrolysis occurs before peptide cleavage, with the former reaction displaying a burst and the latter displaying a lag in product production. The detection of burst kinetics in ATP hydrolysis is indicative of a step after nucleotide hydrolysis being rate-limiting in ATPase turnover. At saturating substrate concentrations, the lag rate constant for peptide cleavage is comparable to thekcatof ATPase, indicating that two hydrolytic processes are coordinated during the first enzyme turnover. The involvement of subunit interaction during enzyme catalysis was detected as positive cooperativity in the binding and hydrolysis of substrates, as well as apparent asymmetry in the ATPase activity in Lon. When our data are taken together, they are consistent with a reaction model in which ATP hydrolysis is used to generate an active enzyme form that hydrolyzes peptide.