Quantitative evaluation of metal ion binding to PvuII restriction endonuclease.
Restriction enzymes are important examples of phosphodiester hydrolysis activity and as such have been of increasing interest to structural biologists. Much of the architecture of endonuclease active sites has been derived from X-ray crystallographic studies. These structures implicate conserved active site acidic residues and the scissile bond of the substrate as coordination ligands of required metal ions. Central to the development of restriction enzyme mechanism is our understanding of the role of metal ion binding in the reaction, an important feature of which is identifying the energetic contributions of the enzyme and the substrate to metal ion affinity. To begin to address this issue, isothermal titration calorimetry (ITC) and 19F NMR spectroscopy have been applied to evaluate metal ion binding by the representative PvuII endonuclease in the absence of substrate. In separate experiments, ITC data demonstrate that PvuII endonuclease binds 2.16 Mn(II) ions and 2.05 Ca(II) metal ions in each monomer active site with Kd values of approximately 1 mM. While neither calorimetry nor protein NMR spectroscopy is directly sensitive to Mg(II) binding to the enzyme, Mn(II) competes with Mg(II) for common sites(s) on PvuII endonuclease. Substitution of the conserved active site carboxylate Glu68 with Ala resulted in a loss of affinity for both equivalents of both Ca(II) and Mn(II). Interestingly, the active site mutant D58A retained an affinity for Mn(II) with Kd approximately 2 mM. Mn(II) paramagnetic broadening in 19F spectra of wild-type and mutant 3-fluorotyrosine PvuII endonucleases are consistent with ITC results. Chemical shift analysis of 3-fluorotyrosine mutant enzymes is consistent with a perturbed conformation for D58A. Therefore, free PvuII endonuclease binds metal ions, and metal ion binding can precede DNA binding. Further, while Glu68 is critical to metal ion binding, Asp58 does not appear to be critical to the binding of at least one metal ion and appears to also have a role in structure. These findings provide impetus for exploring the roles of multiple metal ions in the structure and function of this representative endonuclease.