Yamaguchi T, Yamada R, Tomikawa A, Shudo K, Saito M, Ishikawa F, Saneyoshi M
Department of Biological Sciences, Teikyo University of Science and Technology, Uenohara, Yamanashi, 409-0193, Japan.
Biochem Biophys Res Commun. 2000 Dec 20;279(2):475-81. doi: 10.1006/bbrc.2000.3982.
Telomerase is classified as one of the reverse transcriptases (RTs). To clarify whether l-enantiomers of natural 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the "stretch PCR" method was developed and used for kinetic analysis of the inhibitory effects of these compounds on the enzyme. Among the four l-enantiomers of dNTPs, l-dTTP and l-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver-Burk plot analysis showed that the inhibition modes of l-dTTP and l-dGTP were partially competitive (mixed type) and competitive with the corresponding substrate dNTP, respectively. However, the K(i) values of l-dTTP and l-dGTP (21 and 15 microM) were several times larger than the K(m) values (3-6 microM). These results suggest that the active site of telomerase is not able to discriminate strictly the chirality of dNTPs, although it is more discriminatory than HIV-1 RT.
端粒酶被归类为逆转录酶（RTs）之一。为了阐明天然2'-脱氧核糖核苷5'-三磷酸（dNTPs）的L-对映体是否能被人端粒酶识别，开发了一种基于“延伸PCR”方法的定量端粒酶测定法，并用于动力学分析这些化合物对该酶的抑制作用。在dNTPs的四种L-对映体中，L-dTTP和L-dGTP抑制端粒酶活性，而其他对映体显示出轻微或无抑制作用。Lineweaver-Burk作图分析表明，L-dTTP和L-dGTP的抑制模式分别为部分竞争性（混合型）和与相应底物dNTP竞争性。然而，L-dTTP和L-dGTP的K(i)值（21和15 microM）比K(m)值（3-6 microM）大几倍。这些结果表明，尽管端粒酶的活性位点比HIV-1 RT更具选择性，但它不能严格区分dNTPs的手性。
