Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Chemistry, Biochemistry

Date of Defense

7-17-2008

Graduate Advisor

Cynthia Dupureur, PhD

Committee

Cynthia M. Dupureur

Michael R. Nichols

Chung F. Wong

Keith J. Stine

Abstract

PvuII restriction endonuclease is a homodimeric protein which recognizes and cleaves the palindromic sequence (CAG?CTG) in the presence of Mg(II) ions. Starting with PvuII as a model system, pKa calculations with crystallographically defined metal ligated water are applied to PD?D/ExK motif metallonucleases in order to investigate the activation of nucleophile in metal dependent DNA hydrolysis. These results establish the electrostatic contributions of the metal ions and the conserved Lys in lowering water pKa. The calculated pKa values of metal ligands have been used to simulate the pH dependence of Mg(II) binding to PvuII. The bell shaped pH-rate profile is dissected into three ionizations. One is recognized as from the metal ligands, and the other two have pKa?s similar to calculated metal ligated water pKa in the absence of DNA. The determined pH profiles agree well with previous pH dependence studies on metallonucleases, and the correlation with pKa calculations indicates the direct involvement of metal activated water in catalysis. The different metal occupancies observed in crystal structures lead to controversy regarding the number and function of metal ions involved in DNA hydrolysis by type II restriction endonucleases. Quench flow experiments are used to monitor Mg(II) dependent single and multiple turnover DNA cleavage reactions with PvuII. Several models which differ in order of binding and the number of metal ions supporting catalysis are examined by global fits using DynaFit. The best fitted model has a preference of binding order in the reaction scheme and supports one-metal ion catalysis with 50 fold reduced activity compared with two-metal ion catalysis. The same model is also found to account for multiple turnover data in fits and simulations. A unique reaction scheme for PvuII is established to interpret the determined Mg(II) dependence of kinetic data, which provides an insight into Mg(II) participation in substrate binding, catalysis and product dissociation by restriction endonucleases.

OCLC Number

518020235

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