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 phosp hate groups of the DNA backbone are contacted by the enzyme, To examin e the contribution of backbone contacts to substrate recognition and c atalysis by EcoRV, we exchanged 12 amino acids residues located close to phosphate groups by site-directed mutagenesis. We purified the resu lting EcoRV mutants and characterized them with respect to their DNA b inding and cleavage activity, According to our steady state kinetic an alysis, there are strong interactions between three basic amino acid r esidues (Lys-119, Arg-140, and Arg-226) and the phosphate backbone tha t support specific binding presumably by inducing and maintaining the kinked conformation of the DNA observed in the specific EcoRV DNA comp lex. These contacts are important in both the ground state and the tra nsition state. Other, uncharged residues (Thr-93 and Ser-112), which c ould 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 cat alysis by indirect readout.