SITE-DIRECTED MUTAGENESIS IN THE CATALYTIC CENTER OF THE RESTRICTION-ENDONUCLEASE ECORI

The catalytic center of the restriction endonuclease (ENase) EcoRI is structurally homologous to that of EcoRV, BamHI and PvuII. Each of these ENases contains a short motif of three to four amino acid (aa) residues which are positioned in ct similar orientation to the scissile phosphodiester bond. We have mutated these aa (Pro(90), Asp(91), Glu(111) and Lys(113)) in EcoRI to determine their individual roles in catalysis. The replacement of Asp(91) and Lys(113), respectively, by conservative mutations (Ala(91), Asn(91), Ala(113), Gln(113), His(113) and Leu(113)) resulted in a reduction of binding affinity and complete loss of cleavage activity. Only Lys(113) --> Arg substitution still allows to cleave DNA, albeit with a rate reduced by at least four orders of magnitude. Lys(113) seems to stabilize the structure of the wild-type (wt) ENase since all five ENase variants with mutations at this position show a strongly enhanced tendency to aggregate. The Ala and Gin mutants of Glu(111) bind the recognition sequence slightly stronger than wt EcoRI and cleave it with a low, but detectable rate. Only the Glu(111) --> Lys mutant, in which the charge is reversed, shows neither binding nor cleavage activity. Pro(90) is not important for catalysis, because the Ala(90) mutant cleaves DNA with an only slightly reduced rate. Under star conditions, however, this mutant is even more active than wt EcoRI. Therefore, the charged aa Asp(91), Glu(111) and Lys(113) are essential for catalytic activity of the EcoRI ENase. Differences in the individual contributions of these aa to binding and catalysis, as compared with results obtained with EcoRV and BamHI mutants, show that similar catalytic centers are used in a slightly different way by these three ENases.