A study of the Asp110-Glu112 region of EcoRII restriction endonuclease by site-directed mutagenesis

Site-directed mutagenesis of the ecoRII gene has been used to search for th e active site of the EcoRII restriction endonuclease. Plasmids with point m utations in ecoRII gene resulting in substitutions of amino acid residues i n the Asp110-Glu112 region of the EcoRII endonuclease (Asp110 --> Lys, Asn, Thr, Val, or Ile; Pro111 --> Arg, His, Ala, or Leu; Glu112 --> Lys, Gin, o r Asp) have been constructed. When expressed in E. coli, ail these plasmids displayed EcoRII endonuclease activity, We also constructed a plasmid cont aining a mutant ecoRII gene with deletion of the sequence coding the Gln109 -Pro111 region of the protein. This mutant protein had no EcoRII endonuclea se activity. The data suggest that Asp110, Pro111, and Glu112 residues do n ot participate in the formation of the EcoRII active site. However, this re gion seems to be relevant for the formation of the tertiary structure of th e EcoRII endonuclease.