Dynamics of the Conformational Transitions in the Assembling of the Michaelis Complex of a Bisubstrate Enzyme: A 15N Relaxation Study of Escherichia coli 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase
Dynamics of the Conformational Transitions in the Assembling of the Michaelis Complex of a Bisubstrate Enzyme: A 15N Relaxation Study of Escherichia coli 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase
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DOI：
10.1021/bi8016262
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发表时间：
2009-01-20
期刊：
影响因子：
2.9
通讯作者：
Jin, Changwen
中科院分区：
文献类型：
--
作者：
Lescop, Ewen;Lu, Zhenwei;Jin, Changwen
6-羟甲基-7，8-二氢蝶呤焦磷酸激酶(HPPK)催化焦磷酸盐从ATP向6-羟甲基-7，8-二氢蝶呤(HIP)的转移，遵循一个有序的双动力学机制，ATP首先与酶结合。X射线结晶学表明，HPPK在催化循环过程中发生了显著的构象变化，定点突变、生化和结晶学分析表明，这种构象变化是酶催化所必需的。然而，该酶的动力学性质还没有被实验测量。在这里，我们报道了N-15核磁共振松弛研究了大肠杆菌HPPK从载脂蛋白形式到含镁三磷酸腺苷的二元底物复合体(由三磷酸腺苷类似物代表)到米氏复合体(由镁三磷酸腺苷代表)和幽门螺杆菌(以7，7-二甲基-6-羟基蝶呤代表)的动力学性质。结果表明，核苷酸与HPPK的结合不会引起酶的动力学性质的重大变化。虽然当酶与配体或底物结合时往往更刚性，但HPPK的内部流动性并没有减少，甚至在二元复合体中适度增加，特别是在催化环中。当形成三元络合物时，催化环的内部流动性显著减弱，但仍有一定的流动性。对于三元络合物的组装，可能需要在二元底物络合物的催化环中增强运动。另一方面，三元络合物的催化环可能需要一定程度的迁移率来实现过渡态的最佳稳定，这可能需要瞬时调整和排列催化残基的侧链位置。这种动态行为可能是双底物酶的特征。
6-Hydroxymethyl-7,8-diydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HIP), which follows an ordered bi-bi kinetic mechanism with ATP binding to the enzyme first. HPPK undergoes dramatic conformational changes during its catalytic cycle as revealed by X-ray crystallography, and the conformational changes are essential for the enzymatic catalysis as shown by site-directed mutagenesis and biochemical and crystallographic analysis of the mutants. However, the dynamic properties of the enzyme have not been measured experimentally. Here, we report a N-15 NMR relaxation study of the dynamic properties of Escherichia coli HPPK from the apo form to the binary substrate complex with MgATP (represented by MgAMPCPP, an ATP analogue) to the Michaelis complex (ternary substrate complex) with MgATP (represented by MgAMPCPP) and HP (represented by 7,7-dimethyl-6-hydroxypterin, an HP analogue). The results show that the binding of the nucleotide to HPPK does not cause major changes in the dynamic properties of the enzyme. Whereas enzymes are often more rigid when bound to the ligand or the substrate, the internal mobility of HPPK is not reduced and is even moderately increased in the binary complex, particularly in the catalytic loops. The internal mobility of the catalytic loops is significantly quenched upon the formation of the ternary complex, but some mobility remains. The enhanced motions in the catalytic loops of the binary substrute complex may be required for the assembling of the ternary complex. On the other hand, some degrees of mobility in the catalytic loops of the ternary complex may be required for the optimal stabilization of the transition state, which may need the instantaneous adjustment and alignment of the side-chain positions of catalytic residues. Such dynamic behaviors may be characteristic of bisubstrate enzymes.