Raman difference studies of GDP and GTP binding to c-Harvey ras

J. H. Wang, D. G. Xiao, H. Deng, Martin R. Webb, Robert Callender

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42 Scopus citations


The vibrational spectra of phosphate modes for GDP and GTP bound to the c-Harvey p21(ras) protein have been determined using 18O isotope edited Raman difference spectroscopy. A number of the phosphate stretch frequencies are changed upon GDP/GTP binding to ras, and the results are analyzed by ab initio calculations and through the use of empirical relationships that relate bond orders and bond lengths to vibrational frequencies. Bound GDP is found to be strongly stabilized by its interactions, mostly electrostatic, with the active site Mg2+. Bound GTP also interacts with the active site Mg2+ via its β-phosphate group, as expected on the basis of crystallographic studies of bound GppNp. The angle between the nonbridging P- O bonds of the γ-phosphate of bound GTP increase by about 1-2°compared to its solution value, thus bringing about a geometry that is closer to planar for these bonds as expected for the putative pentacoordinated transition state geometry of the phosphotransfer reaction. Modeling of the interactions at the nucleotide binding site suggests that the water molecule in-line with the P-O bond is positioned to bring about the change in bond angle. Moreover, a weak fifth bond (about 0.03 vu) appears to be formed between it and the γ- phosphorus atom of bound GTP with a concomitant weakening of the O-P bond between the GDP leaving group and the γ-phosphorus atom. Hence, an important role of the active site structure appears to be the strategic positioning of this in-line water. These structural results are consistent with a reaction pathway for GTP hydrolysis in ras of synchronous bond formation between the γ-phosphorus of GTP and the attacking nucleophile and bond breaking between the γ-phosphorus and the GDP leaving group.

Original languageEnglish (US)
Pages (from-to)11106-11116
Number of pages11
Issue number31
StatePublished - Aug 4 1998

ASJC Scopus subject areas

  • Biochemistry


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