The mechanism of GTP hydrolysis by Ras probed by Fourier transform infrared spectroscopy

Abstract

Time-resolved Fourier transform infrared spectroscopy (FTIR) in combination with photo-induced release of 18O-labeled caged nucleotide has been employed to address mechanistic issues of GTP hydrolysis by Ras protein. Infrared spectroscopy of Ras complexes with nitrophenylethyl (NPE)-[α- 18O2]GTP, NPE-[β-18O4]GTP, or NPE-[γ-18O3]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(Gly12)-Mn2+, Ras(Pro12), Ras(Ala12), and Ras(Val12), were obtained and yielded valuable information about structures of GTP or GDP bound to Ras mutants. IR spectra revealed stronger binding of GDP β-PO3/2- 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 [β- nonbridge-18O2, αβ-bridge-18O]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 31P NMR and mass spectrometry.