Zhu C, Johansson M, Permert J, Karlsson A
Division of Clinical Virology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden.
Biochem Pharmacol. 1998 Oct 15;56(8):1035-40. doi: 10.1016/s0006-2952(98)00150-6.
The kinetic properties of recombinant human mitochondrial deoxyguanosine kinase (dGK, EC 2.7.1.113) for 2'-deoxyguanosine and the clinically important nucleoside analogs 2-chloro-2'-deoxyadenosine (CdA), 9-beta-D-arabinofuranosylguanine (araG) and 2',2',-difluorodeoxyguanosine (dFdG) were determined. The Michaelis-Menten kinetic parameters, comparing ATP and UTP as phosphate donors, demonstrated a marked increase in phosphorylation efficiency (VmaxKm) with UTP in comparison with ATP for both CdA and araG. The difluoro analog dFdG was an efficient substrate for recombinant dGK with an apparent Km of 16 microM with ATP as phosphate donor. We compared the kinetic properties of dGK with those of the related enzyme deoxycytidine kinase (dCK, EC 2.7.1.74). Although the purines 2'-deoxyguanosine (dGuo) and 2'-deoxyadenosine are substrates for both dGK and dCK, only CdA among the purine nucleoside analogs tested was an efficient substrate for both dCK and dGK. In competition with dGuo, the most efficient analog for phosphorylation by dGK was araG, as indicated by a lower Ki value than for CdA and dFdG. Of the purine analogs tested as substrates for dCK, only CdA could compete with 2'-deoxycytidine (dCyd). No inhibition of dCK-mediated dCyd phosphorylation was found by either araG or dFdG. In crude cell extract of HeLa and Capan 2 cells, the major CdA phosphorylation was contributed by dCK, while most araG phosphorylation was a result of dGK activity. Our study with pure recombinant enzymes confirms that dGK is mainly responsible for araG and dFdG phosphorylation, whereas dCK is the most important enzyme for activation of CdA and 2',2'-difluorodeoxycytidine (dFdC).
测定了重组人线粒体脱氧鸟苷激酶（dGK，EC 2.7.1.113）对2'-脱氧鸟苷以及临床上重要的核苷类似物2-氯-2'-脱氧腺苷（CdA）、9-β-D-阿拉伯呋喃糖基鸟嘌呤（araG）和2',2'-二氟脱氧鸟苷（dFdG）的动力学性质。米氏动力学参数表明，以ATP和UTP作为磷酸供体时，与ATP相比，UTP使CdA和araG的磷酸化效率（Vmax/Km）显著提高。二氟类似物dFdG是重组dGK的有效底物，以ATP作为磷酸供体时其表观Km为16 μM。我们比较了dGK与相关酶脱氧胞苷激酶（dCK，EC 2.7.1.74）的动力学性质。虽然嘌呤2'-脱氧鸟苷（dGuo）和2'-脱氧腺苷是dGK和dCK的底物，但在所测试的嘌呤核苷类似物中，只有CdA是dCK和dGK的有效底物。在与dGuo竞争时，dGK磷酸化最有效的类似物是araG，其Ki值低于CdA和dFdG。在所测试的作为dCK底物的嘌呤类似物中，只有CdA能与2'-脱氧胞苷（dCyd）竞争。araG或dFdG均未发现对dCK介导的dCyd磷酸化有抑制作用。在HeLa和Capan 2细胞的粗细胞提取物中，CdA的主要磷酸化由dCK贡献，而大多数araG磷酸化是dGK活性的结果。我们对纯重组酶的研究证实，dGK主要负责araG和dFdG的磷酸化，而dCK是激活CdA和2',2'-二氟脱氧胞苷（dFdC）的最重要酶。
