Hydrolytic Cleavage of an RNA-Model Phosphodiester Catalyzed by a Highly Negatively Charged Polyoxomolybdate [Mo7O24]6- Cluster
Hydrolytic Cleavage of an RNA-Model Phosphodiester Catalyzed by a Highly Negatively Charged Polyoxomolybdate [Mo7O24]6- Cluster
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DOI：
10.1021/ja804823g
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发表时间：
2008-12-24
影响因子：
15
通讯作者：
Parac-Vogt, Tatjana N.
中科院分区：
文献类型：
--
作者：
Absillis, Gregory;Cartuyvels, Els;Parac-Vogt, Tatjana N.
利用H-1、P-31和Mo-95 NMR、拉曼和Mo K边扩展X射线吸收精细结构（EXAFS）谱研究了常用的RNA模型底物2-羟丙基-4-硝基苯磷酸（HPNP）在水溶液中的水解。H-1和P-31 NMR光谱表明，在50 ℃和pD 5.9下，HPNP中磷酸二酯键的断裂以6.62 × 10（-6）s（-1）的速率常数进行，得到环状磷酸酯和对硝基苯酚作为唯一的水解产物。NMR光谱没有显示任何顺磁性物质的证据，排除了Mo（VI）还原为Mo（V）的可能性，并表明磷酸二酯键的断裂是纯水解的。Mo K边XANES区域也没有显示在水解反应期间Mo（VI）还原为Mo（V）的任何迹象。k（obs）的pD依赖性表现出钟形曲线，在pD 5.9时观察到最快的裂解。的速率曲线与浓度曲线的多钼酸盐的比较显示了一个惊人的重叠的K-obs曲线与浓度的七钼酸盐，这表明高度带负电荷的[Mo 7 O24]（6-）是水解活性物种。在pD 5.9下，使用固定量的[Mo 7 O24]（6-）和增加量的HPNP进行的动力学实验显示，在25摩尔过量的HPNP下，有轻微的弯曲迹象。数据符合一般的Michaelis-Menten反应方案，允许计算催化速率常数k（2）（3.02 × 10（-4）s（-1））和K-m（1.06 M）。反应混合物的变温P-31 NMR谱显示HPNP P-31共振随温度升高而变宽，这意味着在较高温度下自由和结合HPNP之间的动态交换过程。盐的加入导致HPNP水解的抑制，以及磷酸二甲酯的加入，表明竞争结合[Mo 7 O24]（6-）。在1当量[Mo 7 O24]（6-）的存在下可以实现10当量HPNP的水解，并且多次转换证明该反应是催化的。在不同的水解阶段测量的P-31 NMR和Mo K边EXAFS谱表明，在催化条件下，[Mo_7 O_（24）]（6-）部分转化为[P_2 Mo_5 O_（23）]（6-）。
Hydrolysis of 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP), a commonly used RNA model substrate, was examined in molybdate solutions by means of H-1, P-31, and Mo-95 NMR, Raman, and Mo K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy. H-1 and P-31 NMR spectroscopy indicate that at 50 degrees C and pD 5.9 the cleavage of the phosphodiester bond in HPNP proceeds with a rate constant of 6.62 x 10(-6) s(-1), giving a cyclic phosphate ester and p-nitrophenol as the only products of hydrolysis. The NMR spectra did not show evidence of any paramagnetic species, excluding the possibility of Mo(VI) reduction to Mo(V), and indicating that the cleavage of the phosphodiester bond is purely hydrolytic. The Mo K-edge XANES region also did not show any sign of Mo(VI) to Mo(V) reduction during the hydrolytic reaction. The pD dependence of k(obs) exhibits a bell-shaped profile, with the fastest cleavage observed at pD 5.9. Comparison of the rate profile with the concentration profile of polyoxomolybdates shows a striking overlap of the K-obs profile with the concentration of heptamolybdate, suggesting that the highly negatively charged [Mo7O24](6-) is the hydrolytically active species. Kinetic experiments at pD 5.9 using a fixed amount of [Mo7O24](6-) and increasing amounts of HPNP revealed slight signs of curvature at 25 molar excess of HPNP. The data fit the general Michaelis-Menten reaction scheme, permitting the calculation of the catalytic rate constant k(2) (3.02 x 10(-4) s(-1)) and K-m (1.06 M). Variable temperature P-31 NMR spectra of a reaction mixture revealed broadening of the HPNP P-31 resonance upon increase of temperature, implying the dynamic exchange process between free and bound HPNP at higher temperatures. Addition of salts resulted in the inhibition of HPNP hydrolysis, as well as addition of dimethyl phosphate, suggesting competition for the binding to [Mo7O24](6-). The hydrolysis of 10 equiv of HPNP could be achieved in the presence of 1 equiv of [Mo7O24](6-), and the multiple turnovers demonstrate that the reaction is catalytic. P-31 NMR and Mo K-edge EXAFS spectra measured during different stages of the hydrolysis indicated that under catalytic conditions a partial conversion of [Mo7O24](6-) into [P2Mo5O23](6-) occurs.