Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time.
The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Ras was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (P(3)-1-(2-nitro)phenylethyl guanosine 5'-O-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP x Ras x GDP x AlF(x) transition state analog is known, which represents a "snap shot" along the reaction-pathway. As now revealed, binding of GAP to Ras x GTP shifts negative charge from the gamma to beta phosphate. Such a shift was already identified by FTIR in GTP because of Ras binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP x Ras x GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between beta and gamma phosphate is cleaved. In the intermediate, the released P(i) is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Ras. FTIR analysis reveals the release of P(i) from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein-protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.
Allin
C
C
Lehrstuhl für Biophysik, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Ahmadian
M R
MR
Wittinghofer
A
A
Gerwert
K
K
D002384:Catalysis; D004926:Escherichia coli; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D011905:Genes, ras; D011485:Protein Binding; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D013379:Substrate Specificity
D020690:GTPase-Activating Proteins; D016283:Proto-Oncogene Proteins p21(ras)