Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: Evidence for a loose transition state

A remote labeling method has been developed to determine 180 kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of ¹³C-depleted GTP and [¹⁸O,¹³C]GTP that contains ¹⁸O at phosphoryl positions of mechanistic interest and ¹³C at all carbon positions of the guanosine moiety. Isotope ratios of the nonvolatile substrates and products are measured by using a chemical reaction interface/isotope ratio mass spectrometer. The isotope effects are 1.0012 (0.0026) in the γ nonbridge oxygens, 1.0194 (0.0025) in the leaving group oxygens (the β-γ oxygen and the two β nonbridge oxygens), and 1.0105 (0.0016) in the two β nonbridge oxygens. The KIE in the β-γ bridge oxygen was computed to be 1.0116 or 1.0088 by two different methods. The significant KIE in the leaving group reveals that chemistry is largely rate-limiting whereas the KIEs in the γ nonbridge oxygens and the leaving group indicate a loose transition state that approaches a metaphosphate. The KIE in the two β nonbridge oxygens is roughly equal to that in the β-γ bridge oxygen. This indicates that, in the transition state, R as shifts one-half of the negative charge that arises from P_γ-O_β-γ fission from the β-γ bridge oxygen to the two β nonbridge oxygens. The KIE effects, interpreted in light of structural and spectroscopic data, suggest that Ras promotes a loose transition state by stabilizing negative charge in the β-γ bridge and β nonbridge oxygens of GTP.