Lowe D M, Parmar V, Kemp S D, Larder B A
Department of Molecular Sciences, Wellcome Research Laboratories, Beckenham, Kent, U.K.
FEBS Lett. 1991 May 6;282(2):231-4. doi: 10.1016/0014-5793(91)80484-k.
Two conserved sequence motifs, occurring in HIV-1 reverse transcriptase at residues 110-116 and 183-190, have been studied using site-directed mutagenesis of the cloned gene. In particular, aspartates at positions 185 and 186 have each been mutated to either asparagine or glutamate. The resulting mutant proteins were catalytically inactive but still able to bind the template-primer complex, poly rA-oligo dT. Other mutations in these regions resulted in reduced reverse trascriptase activity but the mutation of tyrosine-183 to serine caused a significant increase in the Km for dTTP and the Ki for inhibition by 3'-azidothymidine-triphosphate, 2',3'-dideoxythymidine-triphosphate and phosphonoformic acid.
利用克隆基因的定点诱变技术，对在HIV-1逆转录酶中位于110-116位和183-190位的两个保守序列基序进行了研究。特别是，185位和186位的天冬氨酸分别被突变为天冬酰胺或谷氨酸。所产生的突变蛋白没有催化活性，但仍能够结合模板-引物复合物，即聚rA-寡聚dT。这些区域的其他突变导致逆转录酶活性降低，但将183位的酪氨酸突变为丝氨酸会使dTTP的Km以及3'-叠氮胸苷三磷酸、2',3'-双脱氧胸苷三磷酸和膦甲酸抑制作用的Ki显著增加。
