Ras catalyzes GTP 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 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-labeled caged GTP. In general, there is excellent agreement with the results of Cepus et al., except in the nu(a)(alpha-PO2-) vibration assignments. The assignments reveal that binding of GTP to Ras induces vibrational uncoupling into mainly individual vibrations of the alpha-, beta-, and 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 different from that in the unbound case. The binding causes an unusual frequency downshift of the GTP beta-PO2- phosphate vibration, whereas the alpha-PO2- and gamma-PO3(2-) phosphate vibrations shift to higher wavenumbers. The frequency 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 changes can be explained by a shift of negative charges from the gamma- to the beta-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 Mg(2+) and at the pro-S beta-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 structural highly conserved phosphate binding loop relative to beta-phosphate therefore seems to be important for the catalysis provided by Ras.
Lehrstuhl für Biophysik, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
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D002384:Catalysis; D020558:GTP Phosphohydrolases / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D010103:Oxygen Isotopes; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D010782:Photolysis; D017550:Spectroscopy, Fourier Transform Infrared; D013237:Stereoisomerism; D013873:Thionucleotides / Q000378:metabolism; D014867:Water; D018631:ras Proteins / Q000378:metabolism*
C057003:1-(2-nitro)phenylethyl-P(3)-guanosine 5'-O-(3-thio)triphosphate; D010103:Oxygen Isotopes; D010710:Phosphates; D013873:Thionucleotides; C034883:guanosine 5'-O-(2-thiotriphosphate); D014867:Water; C061507:P(3)-1-(2-nitro)phenylethylguanosine 5'-O-triphosphate; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D018631:ras Proteins