Abstract
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.
Highlights
From the ‡Biophysics Graduate Program and ¶Department of Chemistry, University of California and the §Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
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
Analysis of the photolysis and hydrolysis Fourier transform infrared spectroscopy (FTIR) spectra of the [-nonbridge-18O2, ␣bridge-18O]GTP isotopomer allowed us to probe for positional isotope exchange
Summary
From the ‡Biophysics Graduate Program and ¶Department of Chemistry, University of California and the §Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720. Evidence supporting an associative mechanism includes the following: (i) an observed linear free energy relationship between the rates of GTP hydrolysis by Ras mutants and the pKa values of the ␥-PO32Ϫ group of GTP bound to these mutants [5]; (ii) the observation that the catalytically important residues interact extensively with the transition state analog GDP-AlF4Ϫ in the crystal structures of Ras-GDP-AlF4Ϫ-GTPase-activating protein [15], Gi␣1GDP-AlF4Ϫ [16], and transducin ␣-GDP-AlF4Ϫ [17] This assumption has been strongly challenged by Maegley et al [4], who argued that the transition state is more likely dissociative in nature based on the following: (i) a wealth of physical organic data that implicates a dissociative, metaphosphate-like transition state in solution reactions of phosphate monoesters, acyl phosphates, phosphorylated amines [6], and phosphoanhydride [18]; and (ii) the fact that the localization of positively charged side chains and metal ions in an enzymatic active site may not change the dissociative transition state of a solution reaction to a more associative one in an enzymatic reaction, as shown for the Escherichia coli alkaline phosphatase [19] and other phosphatases (20 –23). In the case of Ras, two studies employing vibrational spectroscopy have appeared in
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