EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067172-03
Application #
6828280
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Ikeda, Richard A
Project Start
2003-01-01
Project End
2007-12-31
Budget Start
2005-01-01
Budget End
2005-12-31
Support Year
3
Fiscal Year
2005
Total Cost
$229,500
Indirect Cost
Name
Case Western Reserve University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Fishovitz, Jennifer; Li, Min; Frase, Hilary et al. (2011) Active-site-directed chemical tools for profiling mitochondrial Lon protease. ACS Chem Biol 6:781-8
Patterson-Ward, Jessica; Tedesco, Johnathan; Hudak, Jason et al. (2009) Utilization of synthetic peptides to evaluate the importance of substrate interaction at the proteolytic site of Escherichia coli Lon protease. Biochim Biophys Acta 1794:1355-63
Licht, Stuart; Lee, Irene (2008) Resolving individual steps in the operation of ATP-dependent proteolytic molecular machines: from conformational changes to substrate translocation and processivity. Biochemistry 47:3595-605
Lee, Irene; Suzuki, Carolyn K (2008) Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates. Biochim Biophys Acta 1784:727-35
Patterson-Ward, Jessica; Huang, Jon; Lee, Irene (2007) Detection and characterization of two ATP-dependent conformational changes in proteolytically inactive Escherichia coli Lon mutants by stopped flow kinetic techniques. Biochemistry 46:13593-605
Frase, Hilary; Lee, Irene (2007) Peptidyl boronates inhibit Salmonella enterica serovar Typhimurium Lon protease by a competitive ATP-dependent mechanism. Biochemistry 46:6647-57
Vineyard, Diana; Patterson-Ward, Jessica; Lee, Irene (2006) Single-turnover kinetic experiments confirm the existence of high- and low-affinity ATPase sites in Escherichia coli Lon protease. Biochemistry 45:4602-10
Lee, Irene; Berdis, Anthony J; Suzuki, Carolyn K (2006) Recent developments in the mechanistic enzymology of the ATP-dependent Lon protease from Escherichia coli: highlights from kinetic studies. Mol Biosyst 2:477-83
Vineyard, Diana; Zhang, Xuemei; Lee, Irene (2006) Transient kinetic experiments demonstrate the existence of a unique catalytic enzyme form in the peptide-stimulated ATPase mechanism of Escherichia coli Lon protease. Biochemistry 45:11432-43
Frase, Hilary; Hudak, Jason; Lee, Irene (2006) Identification of the proteasome inhibitor MG262 as a potent ATP-dependent inhibitor of the Salmonella enterica serovar Typhimurium Lon protease. Biochemistry 45:8264-74

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