Fritz A, Küster W, Alves J
Institut für Pathologie, GSG-Forschungszentrum für Umwelt und Gesundheit GmbH, Oberschleissheim, Germany.
FEBS Lett. 1998 Oct 30;438(1-2):66-70. doi: 10.1016/s0014-5793(98)01274-5.
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.
限制性内切酶EcoRI的氨基酸残基Asn141被认为与识别序列-GAATTC-中的两个腺嘌呤残基形成三个氢键。我们已将Asn141突变为丙氨酸、天冬氨酸、丝氨酸和酪氨酸。只有丝氨酸突变体在正常缓冲条件下具有活性，尽管其活性比野生型EcoRI低1000倍。丙氨酸和天冬氨酸突变体可被Mn2+激活。在酸性pH值下，后一种突变体在Mn2+存在时比野生型酶更具活性。我们得出结论，Asn141对DNA识别至关重要，丝氨酸可以部分替代它。
