Asp-59 is not important for the catalytic activity of the restriction endonuclease EcoRI

The amino acid Asp-59 was proposed to be involved in EcoRI catalyzed DNA cleavage (Cheng et al., EMBO J. 13, 3927-35, 1994), We have tested this hypothesis by site directed mutagenesis experiments, The four mutants D59A, D59E, D59G, and D59N bind with similar stability to the specific recognition sequence as mild type EcoRI, The D59E mutant cleaves DNA as fast as the wild type enzyme. Specific activities of the other three mutants are five to tenfold lower, Therefore, we conclude that Asp-59 is not involved in catalysis of the EcoRI restriction endonuclease, Consequences for catalytic mechanisms of EcoRI and other restriction enzymes are discussed.