Charulata B. Prasannan
M.S in Chemistry, December 2005, University of Missouri-Saint Louis.
M.Sc. in Chemistry, May, 2002, University of Mumbai, India.
B.Sc. in Chemistry, May, 2000,University of Mumbai, India.

Modulation of Restriction Enzyme PvuII Activity by Metal ion Cofactors

Abstract

Many nucleases require cofactors (usually Mg2+) for activity. Metal ions like Ca2+, Mn2+, Tb3+, Eu3+ etc., can bind at the active site and have varying effects on the enzymatic activity. In a crystal structure with DNA, two Ca2+ ions were bound to the active site of each PvuII monomer, and the metals share the ligands. Ca2+ ions promote DNA binding and not cleavage in many nucleases. We explored the role of Mg2+ and Ca2+ binding at the metal binding sites of PvuII to elucidate the role of each metal site. Ca2+ binding at the catalytic metal site would inhibit the cleavage activity, whereas its binding at the regulatory site can have varied effects. In EcoRV enzyme, it was seen that Ca2+ at the regulatory site increases the activity of the enzyme. We monitored the cleavage kinetics of PvuII in presence of mixed metals. The cleavage kinetics data for Ca2+ and Mg2+ set were modeled using parameters from previous studies on PvuII. Our global analysis on single turnover cleavage kinetics datasets show best fit to a model in which mixed metal species are formed and active. The cleavage rate constants for the mixed metal species ranged from 0.01-0.08 sec-1, which is similar to the rate when only one metal is bound. From earlier work in our lab, Tb3+ was shown to have a tight (2 µM) binding site and a weak binding site in PvuII. The difference in affinity allows one site to be filled with Tb3+ and the other with another metal. Indirect Tb3+ luminescence spectroscopy of the Tb3+ bound to enzyme in presence of other metals indicates that Ca2+ and Mn2+ displace Tb3+ from the enzyme. This was observed by the decrease in the luminescence intensity of E-Tb3+ complex with the addition of Ca2+/Mn2+ ions. Under similar conditions, the addition of Mg2+ ions to the E-Tb3+ complex results in an increase in the signal observed. This indicates the formation of the mixed species E-Tb3+-Mg2+. No enzymatic activity was detected for the enzyme with the addition of Mg2+ to the E-Tb3+ complex, whereas with the addition of Mn2+ ions there was detectable activity. The observed activity with Mn2+ ion was due to the displacement of Tb3+ ions from the active site, forming the active EMn2+Mn2+ species. Although the E-Mg2+-Tb3+ species is catalytically inactive, it does bind the DNA as confirmed by fluorescence anisotropy using nonhydrolyzable phosphoamidate DNA.

Defense of Dissertation Committee