Transition State Analogue Structures of Human Phosphoglycerate Kinase Establish the Importance of Charge Balance in Catalysis
Transition State Analogue Structures of Human Phosphoglycerate Kinase Establish the Importance of Charge Balance in Catalysis
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DOI：
10.1021/ja100974t
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发表时间：
2010-05-12
影响因子：
15
通讯作者：
Waltho, Jonathan P.
中科院分区：
文献类型：
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作者：
Cliff, Matthew J.;Bowler, Matthew W.;Waltho, Jonathan P.
由磷酸甘油酸激酶（PGK）形成的过渡态类似物（TSA）已被用于验证过渡态内电荷平衡主导酶催化磷酸化转移的假设。利用x射线晶体学和基于f -19的核磁共振方法测定了PGK的三氟镁酸盐（MgF3-）和四氟铝酸盐（AlF4-）配合物的高分辨率结构，揭示了这种原型代谢激酶催化相关状态的性质。重要的是，在之前的基态结构中协调a-磷酸基团的K219侧链被隔离成协调金属氟化物，从而创造了一个与转移的磷酰基互补的电荷环境。根据过渡态组织中电荷平衡的优势，取代K219A导致结合的氟化铝的电荷相应减少，转变为三氟铝酸盐（AlF30）配合物。AlF30片段保留了在AlF4- TSA复合物中观察到的八面体几何形状，这支持了一些广泛报道的磷酸基转移酶的三角形AlF30复合物可能被错配并且实际上含有MgF3-的建议。
Transition state analogue (TSA) complexes formed by phosphoglycerate kinase (PGK) have been used to test the hypothesis that balancing of charge within the transition state dominates enzyme-catalyzed phosphoryl transfer. High-resolution structures of trifluoromagnesate (MgF3-) and tetrafluoroaluminate (AlF4-) complexes of PGK have been determined using X-ray crystallography and F-19-based NMR methods, revealing the nature of the catalytically relevant state of this archetypal metabolic kinase. Importantly, the side chain of K219, which coordinates the a-phosphate group in previous ground state structures, is sequestered into coordinating the metal fluoride, thereby creating a charge environment complementary to the transferring phosphoryl group. In line with the dominance of charge balance in transition state organization, the substitution K219A induces a corresponding reduction in charge in the bound aluminum fluoride species, which changes to a trifluoroaluminate (AlF30) complex. The AlF30 moiety retains the octahedral geometry observed within AlF4- TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF30 complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF3-.