Active site contacts in the purine nucleoside phosphorylase--hypoxanthine complex by NMR and ab initio calculations.
Active site contacts in the purine nucleoside phosphorylase--hypoxanthine complex by NMR and ab initio calculations.
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DOI：
10.1021/bi048167i
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发表时间：
2004-12
期刊：
影响因子：
2.9
通讯作者：
H. Deng;S. Cahill;J. Abad;A. Lewandowicz;R. Callender;V. Schramm;Roger A. Jones
中科院分区：
文献类型：
--
作者：
H. Deng;S. Cahill;J. Abad;A. Lewandowicz;R. Callender;V. Schramm;Roger A. Jones
用核磁共振波谱研究了具有特殊(15)N标记的次黄嘌呤(HX)与嘌呤核苷磷酸化酶(PNP)之间的氢键作用。HX以N-7H互变异构体的形式与人PNP结合，N-7H(1)H和(15)N的化学位移分别位于13.9ppm和156.5 ppm，与溶液的值相似。相反，PNP.Hx复合体中N-1H的(1)H和(15)N化学位移在结合时分别向下移动3.5ppm和7.5ppm至15.9ppm和178.8 ppm。因此，络合物中N-1H处的氢键比N-7H处的强。对模拟溶液中HX和PNP结合的模型体系进行了从头算化学位移计算，以解释核磁共振数据。实验中的N-7H化学位移变化是由两个活性中心接触的竞争效应引起的。Glu201到N-1H的氢键导致N-7H基团的前场移动，而局部氢键(从Asn243的C=O到N-7H)导致下场移动。观察到的N-7H化学位移可以用大约0.13A的氢键距离重现(但在实验误差范围内)，这一值与牛PNP.Hx络合物的X射线晶体结构中的实验值相差不大。核磁共振和从头算化学位移计算分析的结合使用为理解抗癌药物靶标PNP中的酶-配体相互作用提供了一种新的方法。这种方法有可能成为结构确定的高分辨率工具。
Hypoxanthine (Hx) with specific (15)N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H (1)H and (15)N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the (1)H and (15)N chemical shifts of N-1H in the PNP.Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance approximately 0.13 A shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP.Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.