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Asp‐59 is not important for the catalytic activity of the restriction endonuclease Eco RI
Author(s) -
Grabowski Gabriele,
Maass Günter,
Alves Jürgen
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00075-0
Subject(s) - ecori , restriction enzyme , mutant , site directed mutagenesis , mutagenesis , dna , endonuclease , microbiology and biotechnology , recognition sequence , biology , chemistry , enzyme , biochemistry , gene
The amino acid Asp‐59 was proposed to be involved in Eco RI 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 wild type Eco RI. 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 Eco RI restriction endonuclease. Consequences for catalytic mechanisms of Eco RI and other restriction enzymes are discussed.