NMR localization of protons in critical enzyme hydrogen bonds.
NMR localization of protons in critical enzyme hydrogen bonds.
复制标题
DOI：
10.1021/ja0728223
复制
发表时间：
2007-07
影响因子：
15
通讯作者：
Shasad Sharif;E. Fogle;M. Toney;G. Denisov;I. Shenderovich;G. Buntkowsky;P. Tolstoy;Monique Chan Huot;H. Limbach
中科院分区：
文献类型：
--
作者：
Shasad Sharif;E. Fogle;M. Toney;G. Denisov;I. Shenderovich;G. Buntkowsky;P. Tolstoy;Monique Chan Huot;H. Limbach
利用15N核磁共振波谱和氢键关联，我们在微晶和水溶液中定位了天冬氨酸氨基转移酶中的一个机械临界质子。它是在Asp222的羧酸盐O和吡哆醛-5‘-磷酸的吡啶氮之间的氢键上。在中性pH值的水中，天冬氨酸和吡啶是未质子化的，但它们在酶中共享一个质子。结果表明，在非质子性极性溶剂中，这种双核碱是酸性−碱相互作用的典型特征。Asp222的活性中心H键有助于酶中吡啶氮的质子化，这被认为是催化活性的先决条件。我们还表明，应该使用非质子极性溶剂而不是水溶液来模拟酶中的酸−碱行为。
Using 15N NMR spectroscopy and hydrogen bond correlations, we have localized a mechanistically critical proton in aspartate aminotransferase in microcrystals and aqueous solution. It is in a H-bond between a carboxylate O of Asp222 and the pyridine nitrogen of pyridoxal-5‘-phosphate. At neutral pH in water, aspartate and pyridine are unprotonated, but they share a proton in the enzyme. It is shown that such a binuclear base is typical for acid−base interactions in aprotic polar solvents. Active site H-bonds to Asp222 assist protonation of the pyridine nitrogen in the enzyme, which is considered a prerequisite for catalytic activity. We also show that acid−base behavior in enzymes should be modeled using aprotic polar solvents rather than aqueous solutions.