Hori N, Doi T, Karaki Y, Kikuchi M, Ikehara M, Ohtsuka E
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Nucleic Acids Res. 1992 Sep 25;20(18):4761-4. doi: 10.1093/nar/20.18.4761.
T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease.
T4核酸内切酶V催化DNA双链中胸腺嘧啶二聚体糖基键的水解以及通过β-消除作用切割3'-磷酸基团。我们之前通过系统诱变（Doi等人，《美国国家科学院院刊》，1992年，即将发表）和X射线晶体学（Morikawa等人，《科学》，256: 523 - 526，1992年）确定了第一个反应（嘧啶二聚体-糖基化酶活性）的催化位点。结果表明，用谷氨酰胺或天冬氨酸取代Glu23会完全消除糖基化酶活性。我们描述了使用22个T4核酸内切酶V突变体以及一个含有无碱基位点的DNA小双链体对第二个反应（脱嘌呤/脱嘧啶内切核酸酶活性）的研究。用谷氨酰胺取代Glu23会消除第二个反应，但用天冬氨酸取代则不会。发现突变体（23 Asp）和野生型的最适pH分别为5.0和5.5。我们得出结论，23位的羧酸根阴离子可能在内切酶的β-消除反应中作为通用碱起作用。
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