Probing the indirect readout of the restriction enzyme EcoRV. Mutational analysis of contacts to the DNA backbone.

According to the crystal structure of the specific EcoRV.DNA complex, not only the functional groups of the nucleobases but also the phosphate groups of the DNA backbone are contacted by the enzyme. To examine the contribution of backbone contacts to substrate recognition and catalysis by EcoRV, we exchanged 12 amino acids residues located close to phosphate groups by site-directed mutagenesis. We purified the resulting EcoRV mutants and characterized them with respect to their DNA binding and cleavage activity. According to our steady state kinetic analysis, there are strong interactions between three basic amino acid residues (Lys-119, Arg-140, and Arg-226) and the phosphate backbone that support specific binding presumably by inducing and maintaining the kinked conformation of the DNA observed in the specific EcoRV.DNA complex. These contacts are important in both the ground state and the transition state. Other, uncharged residues (Thr-93 and Ser-112), which could be involved in hydrogen bonds to the phosphate groups, are needed primarily to stabilize the transition state. An especially important amino acid residue is Thr-37, which seems to couple recognition to catalysis by indirect readout.