(19)F NMR and DFT Analysis Reveal Structural and Electronic Transition State Features for RhoA-Catalyzed GTP Hydrolysis.
(19)F NMR and DFT Analysis Reveal Structural and Electronic Transition State Features for RhoA-Catalyzed GTP Hydrolysis.
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DOI：
10.1002/anie.201509477
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发表时间：
2016-03-01
期刊：
影响因子：
--
通讯作者：
Blackburn GM
中科院分区：
文献类型：
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作者：
Jin Y;Molt RW Jr;Waltho JP;Richards NG;Blackburn GM
RhoA/GAP催化GTP水解为GDP的分子细节知之甚少。我们使用MgF 3 −过渡态类似物（TSA）复合物中的19 F NMR化学位移作为光谱报告子，以指示相应TS中γ-PO 3 −氧的电子分布，这意味着与Mg配位的氧具有最大的电子密度。 通过QM计算验证了这一点，并基于RhoA/GAP-GDP-MgF 3-TSA复合物的结构，给出了水对γ-PO 3-基团进行亲核攻击的过渡态（TS）的电子性质。TS模型显示了一个由20个氢键组成的网络，包括差距Arg 85 ′侧链，但磷酸扭应变和一般碱催化都不明显。亲核水占据了与多基态复合物不同的反应位置，这是由于Arg 85 ′使Gln-63羧酰胺重新取向，从而阻止了水与目标γ-PO 3 −基团的直接氢键结合。
Molecular details for RhoA/GAP catalysis of the hydrolysis of GTP to GDP are poorly understood. We use 19F NMR chemical shifts in the MgF3 − transition state analogue (TSA) complex as a spectroscopic reporter to indicate electron distribution for the γ‐PO3 − oxygens in the corresponding TS, implying that oxygen coordinated to Mg has the greatest electron density. This was validated by QM calculations giving a picture of the electronic properties of the transition state (TS) for nucleophilic attack of water on the γ‐PO3 − group based on the structure of a RhoA/GAP‐GDP‐MgF3 − TSA complex. The TS model displays a network of 20 hydrogen bonds, including the GAP Arg85′ side chain, but neither phosphate torsional strain nor general base catalysis is evident. The nucleophilic water occupies a reactive location different from that in multiple ground state complexes, arising from reorientation of the Gln‐63 carboxamide by Arg85′ to preclude direct hydrogen bonding from water to the target γ‐PO3 − group.