Kinetic Characterization of Lon Protease

在本申请中，我们专注于确定ATP依赖性蛋白酶Lon的动力学细节， 解决两个具体问题：1）ATP结合和水解的时间如何影响催化剂的活性？ 未折叠蛋白质降解的效率，和2）切割位点的底物决定因素是什么？ 由于细胞蛋白质降解的速率取决于ATP依赖性酶的催化效率， 蛋白酶，重要的是研究这些酶如何协调ATP结合和水解， 肽切割以获得最大的蛋白质降解效率。 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水解发生之前，肽裂解，和限速步骤， 肽降解应表现出依赖于ATP水解。由于前稳态动力学技术 允许测定与ATP酶和肽酶反应相关的微观速率常数， 我们将使用这种技术来建立沿着Lon反应途径发生的事件的顺序。 为了深入了解ATP水解和蛋白质水解过程之间的关系，我们将评估 Lon切割含有多个切割位点的多肽底物。此外，我们将确定 Lon切割确定的肽底物的能量需求，所述肽底物采用螺旋构象， 通过评估ATP水解是否是解折叠以及水解所必需的来结合RNA。我们 还将使用以下方法对两种哺乳动物Lon（rot Lon）蛋白酶进行稳态动力学表征： 以合成肽FRETN 89-98为底物，评价E.大肠杆菌和结核分枝杆菌 朗此外，我们将在体外表征人和小鼠Lon对B F 1-ATP酶的降解， 评估mt Lon和F1-ATPase降解之间的功能关系，以了解其作用 在线粒体功能中扮演了重要角色。

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 (rot 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 F 1-ATPase degradation to obtain insight into the role played by Lon in rendering mitochondria function.