Brody R S, Frey P A
Biochemistry. 1981 Mar 3;20(5):1245-52. doi: 10.1021/bi00508a030.
Nucleotidyl transfer catalyzed by DNA polymerase I from Escherichia coli proceeds with greater than 97% inversion of configuration at P alpha of the alpha-phosphorothioate analogue of dATP. This is shown by experiments in which dAMPS,18O2 is stereospecifically phosphorylated to (Sp)-dATP alpha S, alpha 18O2, which is then copolymerized with dTTP by DNA polymerase. The product of the polymerization is degraded to dAMPS,18O by methods that do not affect the configuration of the phosphorothioate. After the dAMPS,18O is stereospecifically phosphorylated, the resulting (Sp)-dATP alpha S, alpha 18O is copolymerized as before with dTTP. The 18O is found in the displaced pyrophosphate by mass spectral analysis and so must have been in the pyrophosphate bridge of (Sp)-dATP alpha S, alpha 18O. Since this 18O was originally non-bridging in (Sp)-dATP alpha S, alpha 18O2, the phosphorothioate configuration must have been inverted in the polymerization reaction. This confirms the determination of P. M. J. Burgers & F. Eckstein [(1979) J. Biol. Chem. 254, 6889-6893], who used kinetic correlations based on the stereoselectivity of snake venom phosphodiesterase to deduce the stereochemistry of this reaction.
大肠杆菌DNA聚合酶I催化的核苷酸转移反应中，dATP的α-硫代磷酸酯类似物的Pα处构型翻转率超过97%。实验表明，dAMPS，18O2被立体特异性磷酸化为（Sp）-dATPαS，α18O2，然后由DNA聚合酶与dTTP共聚。聚合产物通过不影响硫代磷酸酯构型的方法降解为dAMPS，18O。在dAMPS，18O被立体特异性磷酸化后，所得的（Sp）-dATPαS，α18O如前所述与dTTP共聚。通过质谱分析发现18O存在于取代的焦磷酸中，因此它一定存在于（Sp）-dATPαS，α18O的焦磷酸桥中。由于这个18O最初在（Sp）-dATPαS，α18O2中是非桥连的，所以硫代磷酸酯构型在聚合反应中一定发生了翻转。这证实了P. M. J. Burgers和F. Eckstein [(1979) J. Biol. Chem. 254, 6889-6893]的测定结果，他们基于蛇毒磷酸二酯酶的立体选择性利用动力学相关性推断了该反应的立体化学。
