Black M E, Rechtin T M, Drake R R
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, USA.
J Gen Virol. 1996 Jul;77 ( Pt 7):1521-7. doi: 10.1099/0022-1317-77-7-1521.
Despite the extensive use of antiviral drugs for the treatment of herpesvirus infections and as pro-drugs for ablative gene therapy of cancer, little structural information about the drug activating enzyme, herpes simplex virus type 1 thymidine kinase (TK), was available until recently. In the absence of the three-dimensional structure we sought to elucidate the function of the key aspartic acid residue (Dl62) present within a highly conserved tri-peptide motif that is thought to function in nucleoside binding. In this study we generated a mutant, D162Q, by site-directed mutagenesis, purified both the wild-type and mutant TKs to near homogeneity by single-step affinity chromatography and determined the kinetic parameters for thymidine, ATP, dTMP and dTTP interactions. A 12-fold increase in Km for thymidine by D162Q TK (Km = 6.67 microM) relative to wild-type enzyme (Km = 0.56 microM) was observed and the absence of any alteration in Km for ATP suggests that D162 participates in nucleoside binding. Furthermore, the Ki for dTMP is significantly higher for D162Q TK than for HSV-1 TK which is indicative of a shared or overlapping binding site with thymidine. This assessment is further supported by the different inhibition patterns of D162Q and wild-type TKs observed using [alpha-32P]5-N3dUMP photoaffinity labelling in the presence of thymidine, ganciclovir or dTMP. Interestingly, the Ki for dTTP was 30-fold lower for D162Q TK (Ki = 2.2 microM) than for the wild-type enzyme (Ki = 65.8 microM) which provides further evidence of the importance of D162 in TK function.
尽管抗病毒药物被广泛用于治疗疱疹病毒感染以及作为癌症消融基因治疗的前体药物，但直到最近，关于药物激活酶——单纯疱疹病毒1型胸苷激酶（TK）的结构信息仍非常有限。由于缺乏三维结构，我们试图阐明存在于一个高度保守的三肽基序中的关键天冬氨酸残基（D162）的功能，该基序被认为在核苷结合中发挥作用。在本研究中，我们通过定点诱变产生了一个突变体D162Q，通过单步亲和层析将野生型和突变型TK均纯化至接近均一状态，并测定了胸苷、ATP、dTMP和dTTP相互作用的动力学参数。相对于野生型酶（Km = 0.56 microM），观察到D162Q TK对胸苷的Km增加了12倍（Km = 6.67 microM），而ATP的Km没有任何变化，这表明D162参与核苷结合。此外，D162Q TK对dTMP的Ki显著高于单纯疱疹病毒1型TK，这表明其与胸苷存在共享或重叠的结合位点。在胸苷、更昔洛韦或dTMP存在的情况下，使用[α-32P]5-N3dUMP光亲和标记观察到的D162Q和野生型TK不同的抑制模式进一步支持了这一评估。有趣的是，D162Q TK对dTTP的Ki（Ki = 2.2 microM）比野生型酶（Ki = 65.8 microM）低30倍，这进一步证明了D162在TK功能中的重要性。
