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 O-18-labeled caged nucleotide has been emplo yed to address mechanistic issues of GTP hydrolysis by Ras protein. Infrare d spectroscopy of Ras complexes with nitrophenylethyl (NPE)-[alpha-O-18(2)] GTP, NPE-[beta-O-18(4)]GTP, or NPE-[gamma-O-18(3)]GTP upon photolysis or du ring hydrolysis afforded a substantially improved mode assignment of phosph oryl group absorptions. Photolysis spectra of hydroxyphenylacyl-GTP and hyd roxyphenylacyl-GDP bound to Ras and several mutants, Ras(Gly(12))-Mn2+, Ras (Pro(12)), Ras(Ala(12)), and Ras(Val(12)), mere obtained and yielded valuab le information about structures of GTP or GDP bound to has mutants. IR spec tra revealed stronger binding of GDP beta-PO32- 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-nonbrid ge-O-18(2), alpha beta-bridge-O-18]GTP isotopomer allowed us to probe for p ositional isotope exchange. Such a reaction might signal the existence of m etaphosphate as a discrete intermediate, a key species for a dissociative m echanism. No positional isotope exchange was observed. Overall, our results support a concerted mechanism, but the transition state seems to have a co nsiderable amount of dissociative character. This work demonstrates that ti me-resolved FTIR is highly suitable for monitoring positional isotope excha nge and advantageous in many aspects over previously used methods, such as P-31 NMR and mass spectrometry.