Kinetic Characterization of Lon Protease

Lon 蛋白酶的动力学表征

基本信息

批准号：
6560955
负责人：
IRENE LEE
金额：
$ 23.35万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-01-01 至 2007-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6560955
关键词：
Escherichia coli adenosine diphosphate adenosine triphosphate adenosinetriphosphatase chemical kinetics computer simulation endopeptidases enzyme mechanism heat shock proteins hydrolysis matrix assisted laser desorption ionization phosphates protein binding protein degradation proteolysis recombinant proteins

项目摘要

DESCRIPTION (provided by applicant): In this application, we focus on determining the kinetic details of the ATP-dependent protease Lon by addressing two specific questions: 1) how does the timing of ATP binding and hydrolysis affect the catalytic efficiency of unfolded protein degradation, and 2) What are the substrate determinants of the cleavage sites? Since the rate of cellular protein degradation is dependent on the catalytic efficiency of ATP-dependent proteases, it is important to investigate how these enzymes coordinate ATP binding and hydrolysis with peptide cleavage to obtain maximal protein degradation efficiency. Based upon steady-state velocity and product inhibition as well as preliminary pre-steady state kinetic analyses, we propose that ATP hydrolysis occurs prior to peptide cleavage, and the rate-limiting step for peptide degradation should exhibit dependence on ATP hydrolysis. Since pre-steady state kinetic techniques allow one to determine the microscopic rate constants associated with the ATPase and the peptidase reactions, we will employ this technique to establish the sequence of events occurring along the Lon reaction pathway. To gain insight into the relationship between ATP hydrolysis and processive proteolysis, we will evaluate how Lon cleaves polypeptide substrates containing multiple cleavage sites. In addition, we will determine the energetic requirement of Lon cleaving a defined peptide substrate that adopts a helical conformation upon binding to RNA by assessing whether ATP hydrolysis is required for unfolding as well as for hydrolysis. We will also pursue steady-state kinetic characterization of the two mammalian Lon (mt Lon) proteases using the synthetic peptide FRETN 89-98 as substrate to evaluate the mechanistic similarities between E. coli and mt Lon. Furthermore, we will characterize the in vitro degradation of b F 1-ATPase by human and mouse Lon to evaluate the functional relationship between mt Lon and F1-ATPase degradation to obtain insight into the role played by Lon in rendering mitochondria function.

描述（由申请人提供）：在本申请中，我们通过解决两个具体问题来重点确定 ATP 依赖性蛋白酶 Lon 的动力学细节：1）ATP 结合和水解的时间如何影响未折叠蛋白质降解的催化效率，以及 2) 切割位点的底物决定因素是什么？由于细胞蛋白质降解速率取决于 ATP 依赖性蛋白酶的催化效率，因此研究这些酶如何协调 ATP 结合和水解与肽裂解以获得最大蛋白质降解效率非常重要。基于稳态速度和产物抑制以及初步的稳态前动力学分析，我们提出 ATP 水解发生在肽裂解之前，并且肽降解的限速步骤应表现出对 ATP 水解的依赖性。由于预稳态动力学技术允许确定与 ATP 酶和肽酶反应相关的微观速率常数，因此我们将采用该技术来建立沿 Lon 反应途径发生的事件序列。为了深入了解 ATP 水解和持续蛋白水解之间的关系，我们将评估 Lon 如何切割含有多个切割位点的多肽底物。此外，我们将通过评估解折叠和水解是否需要 ATP 水解来确定 Lon 切割特定肽底物的能量需求，该特定肽底物在与 RNA 结合时采用螺旋构象。我们还将使用合成肽 FRETN 89-98 作为底物对两种哺乳动物 Lon (mt Lon) 蛋白酶进行稳态动力学表征，以评估大肠杆菌和 mt Lon 之间的机制相似性。此外，我们将表征人和小鼠 Lon 对 b F 1-ATPase 的体外降解，以评估 mt Lon 和 F1-ATPase 降解之间的功能关系，从而深入了解 Lon 在赋予线粒体功能中所起的作用。