Iwai S, Maeda M, Shirai M, Shimada Y, Osafune T, Murata T, Ohtsuka E
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Biochemistry. 1995 Apr 11;34(14):4601-9. doi: 10.1021/bi00014a013.
The reaction mechanism of bacteriophage T4 endonuclease V was investigated using modified oligodeoxyribonucleotide duplexes containing a cis-syn thymine dimer. For the pyrimidine dimer glycosylase step, the formation of a covalent intermediate has been proposed. A fluorine atom was attached to the 2'-position of the 5'-component of the thymine dimer site, which could stabilize the covalent complex and prevent the ring opening of the sugar moiety. The strand cleavage of the 12 base pair substrate analog did not occur, although the glycosyl bond was cleaved by this enzyme. A covalent enzyme--substrate complex was separated by gel electrophoresis under denaturing conditions. It was shown that the enzyme molecules were completely converted to a stable complex in the reaction mixture. Two mechanisms have been proposed for the beta-elimination step. A 12-mer containing a phosphorothioate linkage between adjacent thymidines was prepared. The diastereomers were separated, and the absolute configurations were determined. After formation of the thymine dimer and 32P-labeling of the 5'-terminus, these oligonucleotides were annealed to the complementary 12-mer, and the reaction rates of the pyrimidine dimer glycosylase step and the overall reaction for each duplex were measured under the substrate-saturation conditions. The rate constants indicated that the chemical reaction at the beta-elimination step was rate-limiting. Since no difference was observed in the rate constants for the Rp- and Sp-phosphorothioate substrates, it is concluded that the beta-elimination reaction is catalyzed, not by the internucleotide phosphate, but by an amino acid residue of the enzyme.
利用含有顺式- syn胸腺嘧啶二聚体的修饰寡脱氧核糖核苷酸双链体研究了噬菌体T4内切核酸酶V的反应机制。对于嘧啶二聚体糖基化酶步骤，有人提出了共价中间体的形成。在胸腺嘧啶二聚体位点的5'-组分的2'-位连接了一个氟原子，它可以稳定共价复合物并防止糖部分的开环。尽管该酶能切割糖苷键，但12碱基对底物类似物的链切割并未发生。在变性条件下通过凝胶电泳分离出共价酶-底物复合物。结果表明，在反应混合物中酶分子完全转化为稳定的复合物。对于β-消除步骤提出了两种机制。制备了在相邻胸苷之间含有硫代磷酸酯键的12聚体。分离出非对映异构体并确定其绝对构型。在形成胸腺嘧啶二聚体并对5'-末端进行32P标记后，将这些寡核苷酸与互补的12聚体退火，并在底物饱和条件下测量每个双链体的嘧啶二聚体糖基化酶步骤的反应速率和整体反应速率。速率常数表明β-消除步骤的化学反应是限速的。由于Rp-和Sp-硫代磷酸酯底物的速率常数没有差异，因此得出结论，β-消除反应不是由核苷酸间磷酸催化的，而是由酶的氨基酸残基催化的。
