Modulation of restriction enzyme PvuII activity by metal ion cofactors.

摘要

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 Ca 2+ 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 microM) 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+/Mn 2+ 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 phosphoramidate DNA.