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

A remote labeling method has been developed to determine (18)O kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of (13)C-depleted GTP and [(18)O,(13)C]GTP that contains (18)O at phosphoryl positions of mechanistic interest and (13)C 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 gamma nonbridge oxygens, 1.0194 (0.0025) in the leaving group oxygens (the beta-gamma oxygen and the two beta nonbridge oxygens), and 1.0105 (0.0016) in the two beta nonbridge oxygens. The KIE in the beta-gamma 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 gamma nonbridge oxygens and the leaving group indicate a loose transition state that approaches a metaphosphate. The KIE in the two beta nonbridge oxygens is roughly equal to that in the beta-gamma bridge oxygen. This indicates that, in the transition state, Ras shifts one-half of the negative charge that arises from P(gamma)-O(beta-gamma) fission from the beta-gamma bridge oxygen to the two beta 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 beta-gamma bridge and beta nonbridge oxygens of GTP.

3H-thymidine is a defective tool with which to measure rates of DNA synthesis.

Metabolic incorporation of 3H-thymidine into cellular DNA is a widely used protocol to monitor rates of DNA synthesis and cell proliferation. However, this radiochemical has also been reported to induce cell-cycle arrest and apoptosis in addition to DNA damage. Using stable isotope-labeled thymidine, we demonstrate that 3H-thymidine induces dose-dependent inhibition of the rate of DNA synthesis. This inhibition occurred within the first round of replication after addition of the radiolabeled tracer and demonstrates the cytotoxic effects of conventional doses of 3H-thymidine (typically greater than or equal to 1 microCi/ml). These results thus show that stable isotope methods are superior to radioisotopes for determining rates of DNA synthesis and cell replication. Because 3H-thymidine perturbs the very process it was employed to study, experiments using 3H-thymidine to monitor DNA synthesis and cell proliferation should be interpreted with caution.