Kinetic Characterization of Lon Protease

描述(申请人提供)：在本申请中，我们通过解决两个具体问题来确定依赖于ATP的蛋白酶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；2)裂解位点的底物决定因素是什么？由于细胞蛋白质的降解速度依赖于依赖于ATP的酶的催化效率，因此研究这些酶如何协调ATP结合和水解肽的裂解以获得最大的蛋白质降解效率是非常重要的。基于稳态速度和产物抑制以及初步的稳态前动力学分析，我们认为，ATP水解发生在多肽裂解之前，多肽降解的限速步骤应该表现出对ATP水解的依赖性。由于稳态前动力学技术允许人们确定与ATPase和肽酶反应相关的微观速率常数，我们将使用该技术来建立沿着Lon反应途径发生的事件的序列。为了深入了解ATP水解酶和蛋白水解酶之间的关系，我们将评估Lon如何裂解包含多个裂解位点的多肽底物。此外，我们将通过评估ATP水解是否需要展开和水解来确定Lon切割定义的多肽底物的能量需求，该底物在与RNA结合时采用螺旋构象。我们还将以合成肽FRETN 89-98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，我们将对人和小鼠Lon对bF1-ATPase的体外降解进行表征，以评估mt Lon和F1-ATPase降解之间的功能关系，以深入了解Lon在实现线粒体功能方面所起的作用。

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.