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Kinetic Characterization of Lon Protease

Lon 蛋白酶的动力学表征

基本信息

批准号：
7169608
负责人：
IRENE LEE
金额：
$ 21.76万
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 2009-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7169608
关键词：
ATP Hydrolysis ATP phosphohydrolase ATP-Dependent Proteases Address Adenylyl Imidodiphosphate Adopted Affect Binding Biological Assay Boxing Cells Chemicals Cleaved cell Computer Simulation Data Dependence Detection Development Endopeptidases Enzymes Escherichia coli Event Exhibits Experimental Models F1-ATPase Fluorescence Fluorescence Anisotropy Fluorescence Resonance Energy Transfer Goals Helix (Snails)Homologous Gene Human Human Activities Hydrolysis In Vitro Kinetics Label Measures Methodology Microscopic Mitochondria Modeling Molecular Molecular Conformation Monitor Mus Nuts Object Attachment Oligopeptides Pathway interactions Peptide Hydrolases Peptides Physiological Process Production Proteins Proteolysis RNA RNA Sequences Radiolabeled Rate Reaction Recombinants Role playing therapy Simulate Site Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Surveys Techniques Testing Time Translations Yeasts analog base chemical cleavage ear helix endopeptidase La genetic analysis inorganic phosphate insight polypeptide programs protein degradation radiotracer synthetic peptide

项目摘要

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.

在这个应用中，我们主要通过以下方式来确定ATP依赖的蛋白酶Lon的动力学细节解决两个具体问题：1)ATP结合和水解的时机如何影响催化未折叠蛋白质降解的效率，以及2)裂解位点的底物决定因素是什么？由于细胞蛋白质降解的速度依赖于依赖于ATP的催化效率蛋白水解酶，重要的是要研究这些酶是如何协调ATP结合和与多肽裂解，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，我们认为，三磷酸腺苷的水解发生在肽的裂解之前，并且限速步骤多肽的降解应该依赖于三磷酸腺苷的水解。由于预稳态动能技术使人们能够确定与ATPase和肽酶反应相关的微观速率常数，我们将使用这项技术来建立沿着Lon反应途径发生的事件的序列。为了深入了解三磷酸腺苷水解酶和蛋白水解酶之间的关系，我们将评估 LON裂解含有多个裂解位点的多肽底物。此外，我们将确定 Lon裂解具有螺旋构象的特定多肽底物的能量需求通过评估ATP水解物是否需要去折叠和水解物与RNA结合。我们还将研究两种哺乳动物Lon(ROT Lon)蛋白酶的稳态动力学特征以合成肽FRETN 89-98为底物评价大肠杆菌与Mt. 朗。此外，我们还将研究人和小鼠Lon to对bF1-ATPase的体外降解评估线粒体Lon和F1-ATPase降解的功能关系，以深入了解其作用朗在呈现线粒体功能中扮演的角色。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Utilization of synthetic peptides to evaluate the importance of substrate interaction at the proteolytic site of Escherichia coli Lon protease.

利用合成肽来评估在大肠杆菌蛋白酶的蛋白水解位点底物相互作用的重要性。

DOI：
10.1016/j.bbapap.2009.02.015
发表时间：
2009-09
期刊：
Biochimica et biophysica acta
影响因子：
0
作者：
Patterson-Ward J;Tedesco J;Hudak J;Fishovitz J;Becker J;Frase H;McNamara K;Lee I
通讯作者：
Lee I

Susceptibility of Snails to Infection with Schistosomes is influenced by Temperature and Expression of Heat Shock Proteins.

蜗牛对血吸虫感染的易感性受温度和热激蛋白表达的影响。

DOI：
10.4172/2161-1165.1000189
发表时间：
2015
期刊：
Epidemiology (Sunnyvale, Calif.)
影响因子：
0
作者：
Knight,Matty;Elhelu,O;Smith,M;Haugen,B;Miller,A;Raghavan,N;Wellman,C;Cousin,C;Dixon,F;Mann,V;Rinaldi,G;Ittiprasert,W;Brindley,PJ
通讯作者：
Brindley,PJ

Transient kinetic experiments demonstrate the existence of a unique catalytic enzyme form in the peptide-stimulated ATPase mechanism of Escherichia coli Lon protease.

瞬时动力学实验证明，在大肠杆菌 Lon 蛋白酶的肽刺激 ATP 酶机制中存在独特的催化酶形式。