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MECHANISTIC CHARACTERIZATION OF THE ATP HYDROLYSIS ACTIVITY OF ESCHERICHIA COLI LON PROTEASE USING KINETIC TECHNIQUES

Vineyard, Diana
http://rave.ohiolink.edu/etdc/view?acc_num=case1164049966

Abstract Details

2007, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Lon is an oligomeric serine protease whose proteolytic activity is activated by ATP hydrolysis. Because the ATPase and protease activities of Lon are inevitably intertwined, a clear understanding of the role of ATP hydrolysis in the enzyme is necessary in elucidating the reaction mechanism of Lon and related ATP-dependent proteases. Lon displays an intrinsic ATPase activity which is stimulated by the presence of peptide or protein substrate. Other purine and pyrimidine triphosphates activate Lon protease activity however ATP is the best activator of peptide cleavage. Additionally peptide stimulates ATP hydrolysis to the highest degree compared to the other nucleotide triphosphates. Collectively these results indicate that ATP is the preferred substrate of Lon. Although each monomeric subunit has an identical sequence, Lon contains two types of ATPase sites that have differing affinities for ATP as well as drastically differing rates of ATP hydrolysis. In this dissertation I primarily utilize pre-steady-state kinetic techniques to monitor the rate constants associated with ATP binding and hydrolysis as well as ADP and inorganic phosphate release from Lon. As stated above both a high- and low-affinity ATPase site exist in Lon which display approximately 20-fold differences in affinity and approximately 1000 fold differences in their rate of ATP hydrolysis. Therefore the roles of the two ATPase sites in Lon catalysis were probed in more detail in order to determine their individual effect on peptidase activity and if they were communicating with one another. On the basis that neither the rate of ATP binding, hydrolysis, nor product release was affected by the presence of peptide substrate but the steady-state turnover number is stimulated by peptide, a unique enzyme form of Lon was proposed to exist. This enzyme form named “F”, is implicated to be generated subsequent to the first round of ATP hydrolysis and catalytically hydrolyzes both peptide and ATP. Taken together, my work demonstrated that the ATPase activity of Lon behaves like a molecular motor which couples the binding and hydrolysis of ATP in a manner similar to those found in other AAA+ proteins to drive protein degradation.
Irene Lee (Advisor)
259 p.
Chemistry, Biochemistry
Lon protease; pre-steady-state enzyme kinetics; ATP hydrolysis; half-site reactivity

Recommended Citations

Citations

  • Vineyard, D. (2007). MECHANISTIC CHARACTERIZATION OF THE ATP HYDROLYSIS ACTIVITY OF ESCHERICHIA COLI LON PROTEASE USING KINETIC TECHNIQUES [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1164049966

    APA Style (7th edition)

  • Vineyard, Diana. MECHANISTIC CHARACTERIZATION OF THE ATP HYDROLYSIS ACTIVITY OF ESCHERICHIA COLI LON PROTEASE USING KINETIC TECHNIQUES. 2007. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1164049966.

    MLA Style (8th edition)

  • Vineyard, Diana. "MECHANISTIC CHARACTERIZATION OF THE ATP HYDROLYSIS ACTIVITY OF ESCHERICHIA COLI LON PROTEASE USING KINETIC TECHNIQUES." Doctoral dissertation, Case Western Reserve University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1164049966

    Chicago Manual of Style (17th edition)

case1164049966
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.