Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, and.
J Biol Chem. 2011 Aug 19;286(33):29146-29157. doi: 10.1074/jbc.M111.250944. Epub 2011 Jun 21.
DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP(i)). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP(i), a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP(i) complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn(2+), larger than Mg(2+), fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.
DNA 聚合酶在焦磷酸（PP(i)）存在的情况下催化与 DNA 模板退火的 DNA 引物的 3'-5'-焦磷酸解。在聚合反应的这种逆转中，DNA 中的脱氧核苷酸被转化为脱氧核苷 5'-三磷酸。基于 PP(i)的两个磷原子之间连接氧的电荷、大小和几何形状，研究了多种化合物以考察它们进行类似于焦磷酸解的反应的能力。我们描述了一种使用焦钒酸盐（VV）的锰介导的焦磷酸解样活性，该活性由噬菌体 T7 的 DNA 聚合酶催化。我们将此反应命名为焦钒酸解。X 射线吸收光谱揭示了聚合酶-VV 复合物中的 Mn-V 距离比聚合酶-PP(i)复合物中的 Mn-P 距离短。这种在活性位点的结构排列解释了 Mn-VV 的酶促激活。我们提出，Mn(2+) 大于 Mg(2+)，适合聚合酶的活性位点，将 VV 结合到聚合酶的活性位点中。我们的结果可能是第一个证明钒可以替代生物过程中磷的文档。
