The time required for water attack at the phosphorus atom of simple phosphodiesters and of DNA
The time required for water attack at the phosphorus atom of simple phosphodiesters and of DNA
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DOI：
10.1073/pnas.0510879103
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发表时间：
2006-03-14
影响因子：
11.1
通讯作者：
Wolfenden, R
中科院分区：
文献类型：
--
作者：
Schroeder, GK;Lad, C;Wolfenden, R
磷酸二酯键，包括那些连接DNA核苷酸的键，高度抵抗自发水解。水对磷酸二酯中磷原子的侵蚀速率仅为人们所知的上限，其基础是磷酸二甲酯阴离子的水解。该反应至少99%是通过C-O裂解进行的，在25℃下，P-O裂解磷酸二酯阴离子的速率上限为10(-15)S(-1)。为了评估P-O裂解磷酸二酯酶(如葡萄球菌核酸酶)所产生的速率增强，我们决定确定简单的磷酸二酯阴离子P-O裂解的速率常数的实际值。在二戊基磷酸酯中，C-O裂解被空间排除，因此只有P-O裂解才能发生水解。高温下的测量表明，二戊基磷酸阴离子在25摄氏度下t(1/2)为30,000,000年的水中发生水解，这表明DNA的核苷酸间连接对磷的水攻击具有抵抗力。这些结果表明，葡萄球菌核酸酶(k(CAT)=95 S(-1))可使磷酸二酯的水解率提高约10(17)倍。据报道，在碱性溶液中，胸苷基-3‘-5’-胸腺嘧啶核苷(TPT)的分解速度是磷酸二氢戊酯的10(5)倍。然而，我们发现TPT和胸腺嘧啶核苷在pH为7和1M KOH中的分解速度和激活参数相似，分解成一组相似的产物。我们推测TPT的分解是由胸腺嘧啶核苷的分解开始的，而不是由磷酸二酯的水解引起的。
Phosphodiester linkages, including those that join the nucleotides of DNA, are highly resistant to spontaneous hydrolysis. The rate of water attack at the phosphorus atom of phosphodiesters is known only as an upper limit, based on the hydrolysis of the dimethyl phosphate anion. That reaction was found to proceed at least 99% by C-O cleavage, at a rate suggesting an upper limit of 10(-15) s(-1) for P-O cleavage of phosphodiester anions at 25 degrees C. To evaluatethe rate enhancement produced by P-O cleaving phosphodiesterases such as staphylococcal nuclease, we decided to establish the actual value of the rate constant for P-O cleavage of a simple phosphodiester anion. In dineopentyl phosphate, C-O cleavage is sterically precluded so that hydrolysis occurs only by P-O cleavage. Measurements at elevated temperatures indicate that the dineopentyl phosphate anion undergoes hydrolysis in water with a t(1/2) Of 30,000,000 years at 25 degrees C, furnishing an indication of the resistance of the internucleotide linkages of DNA to water attack at phosphorus. These results imply that staphylococcal nuclease (k(cat) = 95 s(-1)) enhances the rate of phosphodiester hydrolysis by a factor of approximate to 10(17). In alkaline solution, thymidylyl-3'-5'-thymidine (TpT) has been reported to decompose 10(5)-fold more rapidly than does dineopentyl phosphate. We find however that TpT and thymidine decompose at similar rates and with similar activation parameters, to a similar set of products, at pH 7 and in 1 M KOH. We infer that the decomposition of TpT is initiated by the breakdown of thymidine, not by phosphodiester hydrolysis.