Article thumbnail
Location of Repository

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

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

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 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, Ras 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

Topics: Biological Sciences
Publisher: National Academy of Sciences
Year: 2004
DOI identifier: 10.1073/pnas.0401675101
OAI identifier: oai:pubmedcentral.nih.gov:428437
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://dx.doi.org/10.1073/pnas... (external link)
  • Suggested articles


    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.