Kinetic Characterization of Lon Protease

Lon 蛋白酶的动力学表征

• 批准号: 6693408
• 负责人: IRENE LEE
• 金额: $ 22.95万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 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/6693408
• 关键词: Kinetic; Characterization; Lon; Protease

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，我们专注于通过解决两个具体问题来确定ATP依赖性蛋白酶Lon的动力学细节：1）ATP结合和水解的时机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簇是什么？由于细胞蛋白质降解的速率取决于ATP依赖性蛋白酶的催化效率，因此研究这些酶如何协调ATP结合和水解与肽切割以获得最大的蛋白质降解效率是重要的。基于稳态速度和产物抑制以及初步的预稳态动力学分析，我们提出，ATP水解发生之前肽裂解，和肽降解的限速步骤应表现出依赖于ATP水解。由于前稳态动力学技术允许一个确定与ATP酶和肽酶反应的微观速率常数，我们将采用这种技术来建立事件发生的顺序沿着Lon反应途径。为了深入了解ATP水解和进行性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ATP水解是否需要解折叠以及水解来确定Lon切割在与RNA结合时采用螺旋构象的限定肽底物的能量需求。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，以评估大肠杆菌之间的机制相似性。coli和mt Lon.此外，我们将在体外表征人和小鼠Lon对B F1-ATPase的降解，以评估mt Lon和F1-ATPase降解之间的功能关系，从而深入了解Lon在使线粒体发挥功能中所起的作用。