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

Xinlin Du, Gavin E. Black, Paolo Lecchi, Fred P. Abramson, Stephen R. Sprang

Research output: Contribution to journalArticlepeer-review

Abstract

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 Pγ-Oβ-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.

Original languageEnglish
Pages (from-to)8858-8863
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number24
DOIs
StatePublished - Jun 15 2004

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