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Deciphering the Proteolytic Mechanism of the ATP-Dependent Protease Lon Using Fluorescent Peptides

Ward, Jessica

Abstract Details

2008, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Lon is an ATP-dependent serine protease that degrades damaged, misfolded and certain regulatory proteins in the cell. The enzyme exists as a homo-oligomer with one ATPase domain and one protease domain on each subunit. Using our fluorescent model peptide substrate, γN89-98, as a tool, we have employed steady-state and pre-steady-state kinetic techniques to evaluate the proteolytic mechanism of Lon protease. Steady-state kinetic analysis of the peptidase activity of Lon with nucleotide triphosphates reveals that while nucleotide binding alone is enough to support peptide hydrolysis, the contribution of nucleotide hydrolysis towards the proteolytic activity is significant. Pre-steady-state experiments reveal a lag phase in the peptidase reaction and that ATP hydrolysis occurs before the peptide cleavage event. We propose that the slow step constitues a peptide translocation event that contains two binding events in which the peptide is delivered from the initial peptide binding site to the protease active site. I was able to measure this slow step using proteolytic inactive Lon mutants and a dansylated version of the γN89-98 peptide as well. Taken together, I propose a mechanism for the first round of peptide hydrolysis. Following initial nucleotide and peptide binding, a nucleotide dependent conformational change occurs. Next, upon ATP hydrolysis, the proteolytic site is activated and peptide is delivered to the protease domain where peptide cleavage occurs. In addition to characterizing the kinetic mechanism of Lon protease, I have also utilized multiple peptide substrates to demonstrate the importance of the P3, P1 and P3’ positions in determining substrate specificity and provide evidence that the C-terminus of the γN protein is important for recognition by Lon.
Michael Zagorski (Committee Chair)
Vernon Anderson (Other)
Thomas Gerken (Other)
James Burgess (Other)
Irene Lee (Other)

Recommended Citations

Citations

  • Ward, J. (2008). Deciphering the Proteolytic Mechanism of the ATP-Dependent Protease Lon Using Fluorescent Peptides [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1194311050

    APA Style (7th edition)

  • Ward, Jessica. Deciphering the Proteolytic Mechanism of the ATP-Dependent Protease Lon Using Fluorescent Peptides. 2008. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1194311050.

    MLA Style (8th edition)

  • Ward, Jessica. "Deciphering the Proteolytic Mechanism of the ATP-Dependent Protease Lon Using Fluorescent Peptides." Doctoral dissertation, Case Western Reserve University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1194311050

    Chicago Manual of Style (17th edition)