Kinetic characterization of the peptidase activity of Escherichia coli Lon reveals the mechanistic similarities in ATP-dependent hydrolysis of peptide and protein substrates.
Kinetic characterization of the peptidase activity of Escherichia coli Lon reveals the mechanistic similarities in ATP-dependent hydrolysis of peptide and protein substrates.
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大肠杆菌 Lon 肽酶活性的动力学表征揭示了肽和蛋白质底物的 ATP 依赖性水解机制的相似性。
DOI：
10.1021/bi0255470
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发表时间：
2002
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：
Thomas-Wohlever,Jennifer;Lee,Irene
Lon是一种ATP依赖性蛋白酶，可降解非结构化蛋白质。在这项研究中，我们已经研究了ATP依赖性ofEscherichia coliLon催化水解一个定义的荧光肽称为S3。在ATP或不可水解的ATP类似物AMPPNP的存在下，S3降解的稳态速度分析表明一种顺序机制，并且在ATP的存在下，反应的kcat是7倍。比较ATP与AMPPNP介导的S3水解的前稳态时间过程表明，ATP水解加速了肽的化学裂解之前的缓慢步骤。产物抑制研究表明，ADP对ATP是竞争性的，但对S3底物是非竞争性的。在缺乏S3的情况下，Lon对ADP的亲和力比ATP高10 - 20倍。然而，S3底物使Lon对ADP的亲和力减弱了7 - 19倍，表明该肽也促进了Lon中的ADP/ATP交换，与蛋白质底物类似。水解的肽产物，Pd 1，表现出非竞争性抑制ATP和S3底物。随着S3浓度的增加，Pd 1的Ki变化很小，Pd 1抑制数据支持在ATP或AMPPNP存在下，Lon催化S3水解的异构机制的存在。根据所收集的数据，提出了一个扩展的动力学机制的ATP依赖的肽酶机制的Lon。
Lon is an ATP-dependent protease that degrades unstructured proteins. In this study, we have examined the ATP dependency ofEscherichia coliLon catalyzing the hydrolysis of a defined fluorogenic peptide known as S3. Steady-state velocity analyses of S3 degradation in the presence of ATP, or the nonhydrolyzable ATP analogue AMPPNP, indicate a sequential mechanism, and thekcatof the reaction was 7-fold higher in the presence of ATP. Comparing the pre-steady-state time courses of the ATP- versus AMPPNP-mediated S3 hydrolysis reveals that ATP hydrolysis accelerates a slow step before the chemical cleavage of peptide. Product inhibition studies indicate that ADP is competitive versus ATP but noncompetitive versus the S3 substrate. In the absence of S3, Lon exhibits a 10−20-fold higher affinity for ADP than ATP. However the S3 substrate weakens the affinity of Lon for ADP by 7−19-fold, indicating that this peptide also promotes ADP/ATP exchange in Lon similar to that observed with protein substrates. The hydrolyzed peptide product, Pd1, exhibited noncompetitive inhibition versus both ATP and S3 substrates. Together with the small change in theKiof Pd1 at increasing S3 concentrations, the Pd1 inhibition data support the existence of an isomechanism in Lon catalyzing the hydrolysis of S3 in the presence of ATP or AMPPNP. Upon the basis of the collected data, an extended kinetic mechanism is proposed for the ATP-dependent peptidase mechanism of Lon.