期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 101, 期 24, 页码 8858-8863出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0401675101
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资金
- NIDDK NIH HHS [DK46371, R56 DK046371, R01 DK046371] Funding Source: Medline
A remote labeling method has been developed to determine O-18 kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of C-13-depleted GTP and [O-18, C-13]GTP that contains O-18 at phosphoryl positions of mechanistic interest and C-13 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-Obeta-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.
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