Book of Abstracts: Albany 2007
June 19-23 2007
Use of Both Novel and Classical Approaches to Evaluate the 1 vs. 2 Metal Ion Nuclease Mechanisms
The involvement of 1 vs. 2 metal ions in the hydrolytic mechanism of Mg(II)-dependent nucleases is a surprisingly complex problem that remains actively debated. We are approaching this problem using two parallel strategies. The first involves the evaluation of reaction rates of novel mixed metal-enzyme complexes. Extremely poor DNA cleavage rates for enzyme complexes containing only one equivalent of a metal ion that supports cleavage is consistent with a two metal ion mechanism. We recently established that the restriction enzyme PvuII endonuclease binds two Ln(III) ions with Kds of 2 μM and 100 μM, respectively, and that both contribute to the stabilization of DNA complexes. The fact that Mg(II) (Kd 2 mM) cannot displace lanthanides from the enzyme provides a unique opportunity to measure DNA cleavage rates with mixed metal-enzyme complexes without the complications of exchange or displacement. 20 μM Tb(III) saturates the tight Tb(III) binding site (1) but is insufficient to occupy the weaker Tb(III) site (2). Luminescence spectroscopy is used to demonstrate that the subsequent addition of 10 mM Mg(II) provides occupancy in the second site without displacing the Tb(III) equivalent in the tight site (1). Enzyme-Tb(III)-Mg(II)-DNA complex formation is confirmed via fluorescence anisotropy and nonhydrolyzable phosphoramidates. Only very residual DNA cleavage (< 1%) can be detected with this complex in single turnover cleavage experiments. In a second, parallel approach, we are applying mechanistic modeling to both single turnover and steady state reaction rates collected as a function of Mg(II). Preliminarily, equilibrium and kinetic data fit best to models involving two classes of metal ion binding sites. While there are some caveats, observations from both strategies support the involvement of two Mg(II) ions in the PvuII reaction. This unique combination of classical and novel approaches should prove useful in other metallonuclease systems.
Department of Chemistry & Biochemistry