The mechanism of GTP hydrolysis by Ras probed by Fourier transform infrared spectroscopy.
Time-resolved Fourier transform infrared spectroscopy (FTIR) in combination with photo-induced release of (18)O-labeled caged nucleotide has been employed to address mechanistic issues of GTP hydrolysis by Ras protein. Infrared spectroscopy of Ras complexes with nitrophenylethyl (NPE)-[alpha-(18)O(2)]GTP, NPE-[beta-(18)O(4)]GTP, or NPE-[gamma-(18)O(3)]GTP upon photolysis or during hydrolysis afforded a substantially improved mode assignment of phosphoryl group absorptions. Photolysis spectra of hydroxyphenylacyl-GTP and hydroxyphenylacyl-GDP bound to Ras and several mutants, Ras(Gly(12))-Mn(2+), Ras(Pro(12)), Ras(Ala(12)), and Ras(Val(12)), were obtained and yielded valuable information about structures of GTP or GDP bound to Ras mutants. IR spectra revealed stronger binding of GDP beta-PO(3)(2-) moiety by Ras mutants with higher activity, suggesting that the transition state is largely GDP-like. Analysis of the photolysis and hydrolysis FTIR spectra of the [beta-nonbridge-(18)O(2), alphabeta-bridge-(18)O]GTP isotopomer allowed us to probe for positional isotope exchange. Such a reaction might signal the existence of metaphosphate as a discrete intermediate, a key species for a dissociative mechanism. No positional isotope exchange was observed. Overall, our results support a concerted mechanism, but the transition state seems to have a considerable amount of dissociative character. This work demonstrates that time-resolved FTIR is highly suitable for monitoring positional isotope exchange and advantageous in many aspects over previously used methods, such as (31)P NMR and mass spectrometry.
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Biophysics Graduate Program, University of California, Berkeley 94720, USA.
Frei
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Kim
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D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006868:Hydrolysis; D007834:Lasers; D008956:Models, Chemical; D010103:Oxygen Isotopes; D010782:Photolysis; D017550:Spectroscopy, Fourier Transform Infrared; D013873:Thionucleotides; D018631:ras Proteins / Q000378:metabolism*
D010103:Oxygen Isotopes; D013873:Thionucleotides; C061507:P(3)-1-(2-nitro)phenylethylguanosine 5'-O-triphosphate; D006160:Guanosine Triphosphate; D018631:ras Proteins