EcoRI variant N199H has enhanced specific activity

Abstract

The Asn199 residue of the EcoRI restriction endonuclease has been replaced with other amino acids to investigate whether it mediates nucleotide recognition or catalytic activity. Cassette mutagenesis gave variants of EcoRI: N199D, N199H, N199L, N199R, N199S and N199V. Their relative cleavage rates were found to be in the following order: N199H > EcoRI (wild type; wt) > N199L > N199V > N199S > N199R > N199D. In particular, EcoRI variant N199H showed about a twofold higher specific activity than that of the wt enzyme.