Ras catalyzes CTP hydrolysis by shifting negative charges from gamma- to beta-phosphate as revealed by time-resolved FTIR difference spectroscopy

FTIR difference spectroscopy has been used to determine the molecular GTPas e mechanism of the small GTP binding protein Ras at the atomic level. The r eaction was initiated by the photolysis of caged GTP bound to Ras. The addi tion of catalytic amounts of the GTPase activating protein (GAP) reduces th e measuring time by 2 orders of magnitude but has no influence on the spect ra as compared to the intrinsic reaction. The reduced measuring time improv es the quality of the data significantly as compared to previously publishe d data [Cepus, V., Scheidig, A., Goody, R. S., and Gerwert, K. (1998) Bioch emistry 37, 10263 - 10271]. The phosphate vibrations are assigned using O-1 8-labeled caged GTP. In general, there is excellent agreement with the resu lts of Cepus et al., except in the nu (u)(alpha -PO2-) vibration assignment s. The assignments reveal that binding of GTP to Ras induces vibrational un coupling into mainly individual vibrations of the alpha-, beta-, acid gamma -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 differe nt from that in the unbound case. The binding causes an unusual frequency d ownshift of the GTP beta -PO2- phosphate vibration. whereas the alpha -PO2- and gamma -PO32- phosphate vibrations shift to higher wavenumbers. The fre quency downshift indicates a lowering of the bond order of the nonbridged P -O bonds of the beta -phosphate group of GTP and GDP. The bond order change s can be explained by a shift of negative charges from the gamma- to the be ta -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 beta -oxygen mainly by interaction of Mg2+ and at the pro-S beta -oxygen mainly by interactions o f the backbone NHs of Lys 16, Gly 15, and Val 14. The correct positioning o f the backbone NHs of Lys 16, Gly 15, and Val 14, and especially the Lys 16 side chain, of the structural highly conserved phosphate binding loop rela tive to beta -phosphate therefore seems to be important for the catalysis p rovided by Ras.