G. Grabowski et al., ASP-59 IS NOT IMPORTANT FOR THE CATALYTIC ACTIVITY OF THE RESTRICTION-ENDONUCLEASE ECORI, FEBS letters, 381(1-2), 1996, pp. 106-110
The amino acid Asp-59 was proposed to be involved in EcoRI catalyzed D
NA cleavage (Cheng et al., EMBO J. 13, 3927-35, 1994), We have tested
this hypothesis by site directed mutagenesis experiments, The four mut
ants D59A, D59E, D59G, and D59N bind with similar stability to the spe
cific 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 endonucle
ase, Consequences for catalytic mechanisms of EcoRI and other restrict
ion enzymes are discussed.