title: Asn141 is essential for DNA recognition by EcoRI restriction endonuclease.
abstract: 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.
authors.affiliation: Institut für Pathologie, GSG-Forschungszentrum für Umwelt und Gesundheit GmbH, Oberschleissheim, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Fritz
authors.forename: W
authors.initials: W
authors.lastname: Küster
authors.forename: J
authors.initials: J
authors.lastname: Alves
mesh_terms: D000225:Adenine / Q000378:metabolism; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D010582:Bacteriophage lambda; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D004279:DNA, Viral; D001216:Asparagine; D004247:DNA; D015246:Deoxyribonuclease EcoRI; D000225:Adenine