Asn141 is essential for DNA recognition by EcoRI restriction endonuclease

The amino acid residue Asn141 of the restriction endonuclease EcoRI was proposed to make three hydrogen bonds to both adenine residues within the recognition sequence -GAATTC-. We have mutated Asn141 to alanine, aspartate, serine, and tyrosine. Only the serine mutant is active under normal buffer conditions although 1000-fold less than wild-type EcoRI. The alanine and aspartate mutants can be activated by Mn2+. At acidic pH the latter mutant becomes even more active than the wild-type enzyme in the presence of Mn2+. We conclude that Asn141 is essential for DNA recognition and that serine can partly substitute it.