DNA cleavage by the EcoRV restriction endonuclease: pH dependence and proton transfers in catalysis

To characterise the pH dependence of phosphodiester hydrolysis by the EcoRV endonuclease in the presence of Mn2+, single turnover reactions on a 12 bp DNA substrate were examined by stopped-flow and quench-flow methods between pH 6.0 and 8.5. At each pH value, the apparent rate constants for phosphodiester hydrolysis increased hyperbolically with the concentration of MnCl2, thus allowing values to be determined for the intrinsic rate constant at saturation with Mn2+ and the equilibrium dissociation constant for Mn2+. The equilibrium constants showed no systematic variation across the pH range tested, while the rate constants increased steeply with increasing pH up to an asymptote above pH 7.5. At low pH conditions, the gradient of a plot of log (rate constant) against pH approached a value of 2. DNA cleavage by EcoRV thus requires the de-protonation of two acidic groups. To determine whether aspartate 36 is one of the groups, mutants of EcoRV were made with other amino acid residues at position 36. Glutamate caused a partial loss of activity, while all other replacements gave near-zero activities. In contrast to wild-type EcoRV, the mutant with glutamate required the de-protonation of only one acidic group for DNA cleavage. A mechanism for EcoRV is proposed in which the water molecule that hydrolyses the phosphodiester bond is de-protonated by two Bronsted bases, probably the ionised forms of aspartate 36 and glutamate 45.