Needels M C, Fried S R, Love R, Rosenberg J M, Boyer H W, Greene P J
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Proc Natl Acad Sci U S A. 1989 May;86(10):3579-83. doi: 10.1073/pnas.86.10.3579.
The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination. Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases were assessed for activities in vivo and in vitro. The mutant endonuclease with lysine at position 200 exhibits the most in vivo activity of all the position 200 mutants, although the in vitro activity is less than 1/100th of wild-type activity. Five other mutants show more drastically reduced levels of in vivo activity (Cys, Pro, Val, Ser, and Trp). The Cys, Val, and Ser mutant enzymes appear to have in vivo activity which is specific for the wild-type canonical site despite the loss of hydrogen bonding potential at position 200. The Pro and Trp mutants retain in vivo activity which is independent of the presence of the EcoRI methylase. In crude cell lysates, only the Cys mutant shows a very low level of in vitro activity. None of the mutant enzymes show a preference for alternative sites in assays in vitro. The implications of these results are discussed.
人们认为，EcoRI核酸内切酶第200位的精氨酸与序列GAATTC中的鸟嘌呤形成两个氢键，因此是序列识别的重要决定因素。将精氨酸-200替换为其他19种天然存在的氨基酸，并对突变型核酸内切酶进行体内和体外活性评估。第200位为赖氨酸的突变型核酸内切酶在所有第200位突变体中表现出最高的体内活性，尽管其体外活性不到野生型活性的1/100。其他五个突变体的体内活性水平大幅降低（半胱氨酸、脯氨酸、缬氨酸、丝氨酸和色氨酸）。半胱氨酸、缬氨酸和丝氨酸突变酶似乎具有对野生型经典位点具有特异性的体内活性，尽管第200位失去了氢键形成潜力。脯氨酸和色氨酸突变体保留了与EcoRI甲基化酶存在无关的体内活性。在粗细胞裂解物中，只有半胱氨酸突变体显示出非常低水平的体外活性。在体外试验中，没有一种突变酶表现出对替代位点的偏好。讨论了这些结果的意义。
