Gupta A P, Benkovic S J
Biochemistry. 1984 Nov 20;23(24):5874-81. doi: 10.1021/bi00319a029.
(Sp)-2'-Deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate has been synthesized by desulfurization of (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1,1-18O2]diphosphate) with N-bromosuccinimide in [17O]water, followed by phosphorylation with phosphoenolpyruvate-pyruvate kinase. A careful characterization of the product using high-resolution 31P NMR revealed that the desulfurization reaction proceeded with approximately 88% direct in-line attack at the alpha-phosphorus and 12% participation by the beta-phosphate to form a cyclic alpha,beta-diphosphate. The latter intermediate underwent hydrolysis by a predominant nucleophilic attack on the beta-phosphate. This complexity of the desulfurization reaction, however, does not affect the stereochemical integrity of the product but rather causes a minor dilution with nonchiral species. The usefulness of the (Sp)-2'-deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate in determining the stereochemical course of deoxyribonucleotidyl-transfer enzymes is demonstrated by using it to delineate the stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. Upon incubation of this oxygen-chiral substrate with Klenow fragment of DNA polymerase I in the presence of poly[d(A-T)] and Mg2+, a quantitative conversion into 2'-deoxyadenosine 5'-O-[16O,17O,18O]monophosphate was observed. The stereochemistry of this product was determined to be Rp. Since the overall template-primer-dependent conversion of a deoxynucleoside triphosphate into the deoxynucleoside monophosphate involves incorporation into the polymer followed by excision by the 3'----5'-exonuclease activity and since the stereochemical course of the incorporation reaction is known to be inversion, it can be concluded that the stereochemical course of the 3'----5'-exonuclease is also inversion.
(Sp)-2'-脱氧腺苷5'-O-[1-¹⁷O,1-¹⁸O,1,2-¹⁸O]三磷酸已通过在[¹⁷O]水中用N-溴代琥珀酰亚胺对(Sp)-2'-脱氧腺苷5'-O-(1-硫代[1,1-¹⁸O₂]二磷酸)进行脱硫，随后用磷酸烯醇丙酮酸-丙酮酸激酶进行磷酸化反应合成。使用高分辨率³¹P NMR对产物进行仔细表征表明，脱硫反应以约88%的直接直线攻击α-磷和12%的β-磷酸参与形成环状α,β-二磷酸的方式进行。后者的中间体通过对β-磷酸的主要亲核攻击发生水解。然而，脱硫反应的这种复杂性并不影响产物的立体化学完整性，而是导致与非手性物种的轻微稀释。通过用(Sp)-2'-脱氧腺苷5'-O-[1-¹⁷O,1-¹⁸O,1,2-¹⁸O]三磷酸来描绘DNA聚合酶I的3'→5'-外切核酸酶活性的立体化学过程，证明了其在确定脱氧核糖核苷酸转移酶的立体化学过程中的有用性。在聚[d(A-T)]和Mg²⁺存在下，将这种氧手性底物与DNA聚合酶I的Klenow片段一起孵育后，观察到定量转化为2'-脱氧腺苷5'-O-[¹⁶O,¹⁷O,¹⁸O]单磷酸。该产物的立体化学被确定为Rp。由于脱氧核苷三磷酸向脱氧核苷单磷酸的总体模板-引物依赖性转化涉及掺入聚合物，随后通过3'→5'-外切核酸酶活性切除，并且由于已知掺入反应的立体化学过程是翻转，因此可以得出结论，3'→5'-外切核酸酶的立体化学过程也是翻转。
