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

FEBS Letters

Grabowski, Gabriele¹  Maass, Günter¹  Alves, Jürgen¹ 

[1] Zentrum Biochemie, Institut für Biophysikalische Chemie, OE 4350, Medizinische Hochschule Hannover, D-30623 Hannover, Germany

关键词: Restriction enzyme;    Catalysis;    Protein-DNA interaction;    Mg2+ coordination;   

DOI  :  10.1016/0014-5793(96)00075-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 wild 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.

【 授权许可】

Unknown