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首页> 外文期刊>Biochemistry >Ras Catalyzes GTP Hydrolysis by Shifting Negative Charges from #gamma#-to #beta#-Phosphate As Revealed by Time-Resolved FTIR Difference Spectroscopy
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Ras Catalyzes GTP Hydrolysis by Shifting Negative Charges from #gamma#-to #beta#-Phosphate As Revealed by Time-Resolved FTIR Difference Spectroscopy

机译：Ras通过将负电荷从＃gamma＃-磷酸盐转变为＃beta＃-磷酸盐来催化GTP水解，正如时间分辨FTIR差光谱法所揭示的那样

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摘要

FfIR difference spectroscopy has been used to detennine the molecular GTPase mechanism of the small GTP binding protein Ras at the atomic level. The reaction was initiated by the photolysis of caged GTP bound to Ras. The addition of catalytic amounts of the GTPase activating protein (GAP) reduces the measuring time by 2 orders of magnitude but has no influence on the spectra as compared to the intrinsic reaction. The reduced measuring time improves the quality of the data significantly as compared to previously published data [Cepus, V., Scheidig, A., Goody, R. S., and Gerwert, K. (1998) Biochemistry 37, 10263-10271]. The phosphate vibrations are assigned using 18O-1abeled cagedGTP. In general, there is excellent agreement with the results of Cepus et al., except in the Va?l-PO2-) vibration assignments. The assignments reveal that binding of GTP to Ras induces vibrational uncoupling into mainly individual vibrations of the a-, {3-, and y-phosphate groups. In contrast, for unbound GTP, the phosphate vibrations are highly coupled and the corresponding absorption bands are broader. This result indicates that binding to Ras forces the flexible GTP molecule into a strained conformation and induces a specific charge distribution different from that in the unbound case. The binding causes an unusual frequency downshift of the GTP {3-PO2- phosphate vibration, whereas the (l-PO2- and y-PO32- phosphate vibrations shift to higher wavenumbers. The frequency downshift indicates a lowering of the bond order of the nonbridged P-O bonds of the {3-phosphate group ofGTP and GDP. The bond order changes can be explained by a shift of negative charges from the y- to the {3-oxygens. Thereby, the GTP charge distribution becomes more like that in GDP. The charge shift appears to be a key factor contributing to catalysis by Ras in addition to the correct positioning of the attacking water. Ras appears to increase the negative charge at the pro-R {3-oxygen mainly by interaction of Mg2+ and at the pro-S {3-oxygen mainly by interactions of the backbone NHs of Lys 16, Gly 15, and Val 14. The correct positioning of the backbone NHs of Lys 16, Gly 15, and Val 14, and especially the Lys 16 side chain, of the struct61ral highly conserved phosphate binding loop relative to {3-phosphate therefore seems to be important for the catalysis provided bv Ras.
机译：FfIR差异光谱已用于确定小GTP结合蛋白Ras在原子水平上的分子GTPase机制。该反应通过与Ras结合的笼状GTP的光解而引发。催化量的GTPase活化蛋白（GAP）的添加将测量时间减少了2个数量级，但与固有反应相比，对光谱没有影响。与先前公布的数据相比，减少的测量时间显着改善了数据质量[Cepus，V.，Scheidig，A.，Goody，R. S.，and Gerwert，K.（1998）Biochemistry 37，10263-10271]。使用18O-1笼式GTP分配磷酸盐的振动。通常，除了Va？l-PO2-）振动分配外，与Cepus等人的结果非常吻合。研究表明，GTP与Ras的结合导致振动解偶联，主要分解为a-，{3-和y-磷酸盐基团的单个振动。相反，对于未结合的GTP，磷酸盐的振动高度耦合，相应的吸收带更宽。该结果表明与Ras的结合迫使柔性GTP分子变成应变的构象，并诱导不同于未结合情况的比电荷分布。结合会导致GTP {3-PO2-磷酸盐振动发生异常的频率下移，而（1-PO2-和y-PO32-磷酸盐振动则向较高的波数移动。频率下移表示非桥键的键序降低GTP和GDP的{3-磷酸基团的PO键。键顺序的变化可以用负电荷从y-到{3-氧的移动来解释。因此，GTP的电荷分布变得更像GDP。除攻击水的正确位置外，电荷转移似乎是导致Ras催化的关键因素，Ras似乎主要通过Mg2 +和脯氨酸的相互作用增加pro-R {3-氧的负电荷。 -S {3-氧主要是通过Lys 16，Gly 15和Val 14的主链NH相互作用。Lys16，Gly 15和Val 14的主链NH的正确定位，尤其是Lys 16侧链，相对于{3-p因此，对于Ras提供的催化作用，亚硫酸盐似乎很重要。

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