Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution inreal time

The molecular reaction mechanism of the GTPase-activating protein (GAP)-cat alyzed GTP hydrolysis by Pas was investigated by time resolved Fourier tran sform infrared (FTIR) difference spectroscopy using caged GTP (P-3-1-(2-nit ro)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 th e GAP.Ras.GDP.AIF(X) transition state analog is known, which represents a " snap shot" along the reaction-pathway. As now revealed, binding of GAP to R as GTP shifts negative charge from the gamma to beta phosphate. Such a shif t was already identified by FTIR in GTP because of Pas binding and is now s hown to be enhanced by GAP binding. Because the charge distribution of the GAP.Ras.GTP complex thus resembles a more dissociative-like transition stat e and is more like that in GDP, the activation free energy is reduced. An i ntermediate is observed on the reaction pathway that appears when the bond between beta and gamma phosphate is cleaved. In the intermediate, the relea sed P-i is strongly bound to the protein and surprisingly shows bands typic al of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Pas. FTIR analysis reveals the release of P-i from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The appro ach presented allows the study not only of single proteins but of protein-p rotein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.