Time-resolved FTIR studies of the GTPase reaction of H-ras p21 reveal a key role for the β-phosphate
Time-resolved FTIR studies of the GTPase reaction of H-ras p21 reveal a key role for the β-phosphate
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DOI：
10.1021/bi973183j
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发表时间：
1998-07-14
期刊：
影响因子：
2.9
通讯作者：
Gerwert, K
中科院分区：
文献类型：
--
作者：
Cepus, V;Scheidig, AJ;Gerwert, K
本文建立了一种新的红外差谱技术，以原子分辨率的时间分辨模式研究了H-rasp 21（Ras）的GTdR反应，无需结晶。使用位点特异性O-18标记的笼状GTP同位素异构体分析磷酸盐振动。每个核苷酸的一个非桥氧被磷酸链的α-，β-或γ-位置的O-18同位素取代。在光解实验与自由笼GTP，强烈的振动耦合之间观察到所有磷酸基团。Ras笼状GTP的光解和随后的Ras GTP水解反应的研究表明，与游离GTP相比，磷酸振动在很大程度上通过与蛋白质的相互作用而解耦。特征同位素位移允许带分配到孤立的α-，β-和γ-磷酸振动笼GTP，GTP和释放的无机磷酸盐。与游离GTP相比，Ras结合GTP的β（PO 2-）振动频率异常低，表明P-O键级大幅降低。键级下降表明，β（PO 2-）基团的氧原子与蛋白质环境的相互作用比γ-氧原子强得多。由此，电子从β-磷中撤出，并因此也从β/γ-桥接氧中撤出。这导致部分键断裂或至少减弱β/γ-桥接氧和γ-磷原子之间的键，作为GTP水解的推定早期步骤。基于这些结果，我们提出了一个关键作用的β-磷酸GTP水解。磷酸盐谱带的归属为进一步的时间分辨FTIR研究Ras的GTdR反应提供了一个重要的标记。
FTIR difference spectroscopy has been established as a new tool to study the GTPase reaction of H-ras p21 (Ras) in a time-resolved mode at atomic resolution without crystallization. The phosphate vibrations were analyzed using site specifically O-18-labeled caged GTP isotopomers. One nonbridging oxygen per nucleotide was replaced for an O-18 isotope in the alpha-,beta-, or gamma-position of the phosphate chain. In photolysis experiments with free caged GTP, strong vibrational coupling was observed among all phosphate groups. The investigation of Ras.caged GTP photolysis and the subsequent hydrolysis reaction of Ras.GTP showed that the phosphate vibrations are largely decoupled by interaction with the protein in contrast to free GTP. The characteristic isotope shifts allow band assignments to isolated alpha-, beta-, and gamma-phosphate vibrations of caged GTP, GTP, and the liberated inorganic phosphate. The unusually low frequency of the beta (PO2-) vibration of Ras-bound GTP, as compared to free GTP, indicates a large decrease in the P-O bond order. The bond order decrease reveals that the oxygen atoms of the beta (PO2-) group interact much more strongly with the protein environment than the gamma-oxygen atoms. Thereby, electrons are withdrawn from the beta-phosphorus, and thus also from the beta/gamma-bridging oxygen, This leads to partial bond breakage or at least weakening of the bond between the beta/gamma-bridging oxygen and the gamma-phosphorus atom as a putative early step of the GTP hydrolysis. Based on these results, we propose a key role of the beta-phosphate for GTP hydrolysis. The assignments of phosphate bands provide a crucial marker for further time-resolved FTIR studies of the GTPase reaction of Ras.