Document Type
Article
Publication Title
Proceedings of the National Academy of Sciences of the United States of America
Publication Date
6-15-2004
Volume
101
Issue
24
Disciplines
Biology | Life Sciences
Abstract
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.
DOI
10.1073/pnas.0401675101
Rights
© 2004 by The National Academy of Sciences of the USA
Recommended Citation
Du, Xinlin; Black, Gavin E.; Lecchi, Paolo; Abramson, Fred P.; and Sprang, Stephen R., "Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: evidence for a loose transition state" (2004). Biological Sciences Faculty Publications. 513.
https://scholarworks.umt.edu/biosci_pubs/513