Effects of divalent metal ions on the activity and conformation of native and 3‐fluorotyrosine‐ Pvu II endonucleases

The activities of restriction enzymes are important examples of Mg(II)‐dependent hydrolysis of DNA. While a number of crystallographic studies of enzyme–DNA complexes have also involved metal ions, there have been no solution studies exploring the relationship between enzyme conformation and metal‐ion binding in restriction enzymes. Using Pvu II restriction endonuclease as a model system, we have successfully developed biosynthetic fluorination and NMR spectroscopy as a solution probe of restriction‐enzyme conformation. The utility of this method is demonstrated with a study of metal‐ion binding by Pvu II endonuclease. Replacement of 74% (± 10%) of the Tyr residues in Pvu II endonuclease by 3‐fluorotyrosine produces an enzyme with Mg(II)‐supported specific activity and sequence specificity that is indistinguishable from that of the native enzyme. Mn(II) supports residual activity of both the native and fluorinated enzymes; Ca(II) does not support activity in either enzyme, a result consistent with previous studies. 1 H‐ and 19 F‐NMR spectroscopic studies reveal that while Mg(II) does not alter the enzyme conformation, the paramagnetic Mn(II) produces both short‐range spectral broadening and longer range changes in chemical shift. Most interestingly, Ca(II) binding perturbs a larger number of different resonances than Mn(II). Coupled with earlier mutagenesis studies that place Ca(II) in the active site [Nastri, H.G., Evans, P.D., Walker, I.H. & Riggs, P.D. (1997) J. Biol. Chem. 272 , 25761–25767], these data suggest that the enzyme makes conformational adjustments to accommodate the distinct geometric preferences of Ca(II) and may play a role in the inability of this metal ion to support activity in restriction enzymes.