Mechanism of the conformational transitions in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by NMR spectroscopy
Mechanism of the conformational transitions in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by NMR spectroscopy
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DOI：
10.1021/bi061057m
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发表时间：
2006-10-17
期刊：
影响因子：
2.9
通讯作者：
Yan, Honggao
中科院分区：
文献类型：
--
作者：
Li, Guangyu;Felczak, Krzysztof;Yan, Honggao
6-羟甲基-7，8-二氢蝶呤焦磷酸激酶(HPPK)催化焦磷酸从ATP转移到6-羟甲基-7，8-二氢蝶呤(HP)，导致叶酸辅因子的生物合成。HPPK在其催化循环中经历了剧烈的构象变化，这种构象变化是酶催化所必需的。因此，该酶不仅是开发抗菌药物的一个有吸引力的靶标，而且是研究酶焦磷酸转移的催化机理以及蛋白质动力学在酶催化中的作用的一个很好的模型系统。在本研究中，我们报道了二元络合物HPPK中心点MgAMPCPP和三元络合物HPPK中心点MgAMPCPP中心点DMHP的核磁共振溶液结构，其中α，ss-亚甲基三磷酸(AMPCPP)和7，7-二甲基-6-羟基蝶呤(DMHP)分别是底物ATP和HP的类似物。结果表明，HPPK二元络合物的三个催化环可以在缓慢的交换中呈现多种构象，环2和环3中几个残基的多组核磁共振信号以及环1和环3中几个残基的很弱或缺失的NH交叉峰证明了这一点。然而，三元络合物只有一组核磁共振信号，并且交叉峰强度相当均匀，这表明第二底物的结合将二元络合物的多重构象转变为明显单一的三元络合物构象。HPPK核心点MGAMPCPP的核磁共振行为和构象与HPPK的MGSS，γ-亚甲基腺苷三磷酸(MGAMPPCP)络合物的核磁共振行为和构象明显不同。由于在结合亲和力和结合构象方面，MgAMPCPP都是一种较好的HPPK的类似物，因此建议用HPPK中心点的构象性质来代表HPPK的二元底物复合体的构象性质。
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. HPPK undergoes dramatic conformational changes during its catalytic cycle, and the conformational changes are essential for enzymatic catalysis. Thus, the enzyme is not only an attractive target for developing antimicrobial agents but also an excellent model system for studying the catalytic mechanism of enzymatic pyrophosphoryl transfer as well as the role of protein dynamics in enzymatic catalysis. In the present study, we report the NMR solution structures of the binary complex HPPK center dot MgAMPCPP and the ternary complex HPPK center dot MgAMPCPP center dot DMHP, where alpha,ss-methyleneadenosine triphosphate (AMPCPP) and 7, 7-dimethyl-6-hydroxypterin (DMHP) are the analogues of the substrates ATP and HP, respectively. The results suggest that the three catalytic loops of the binary complex of HPPK can assume multiple conformations in slow exchanges as evidenced by multiple sets of NMR signals for several residues in loops 2 and 3 and the very weak or missing NH cross-peaks for several residues in loops 1 and 3. However, the ternary complex shows only one set of NMR signals, and the cross-peak intensities are rather uniform, suggesting that the binding of the second substrate shifts the multiple conformations of the binary complex to an apparently single conformation of the ternary complex. The NMR behaviors and conformations of the binary complex HPPK center dot MgAMPCPP are significantly different from those of HPPK in complex with Mg ss,gamma-methyleneadenosine triphosphate (MgAMPPCP). It is suggested that the conformational properties of the binary substrate complex HPPK center dot MgATP be represented by those of HPPK center dot MgAMPCPP, because MgAMPCPP is a better MgATP analogue for HPPK with respect to both binding affinity and bound conformation.