Vibrational structure of GDP and GTP bound to RAS: an isotope-edited FTIR study.
A complete vibrational description of the bonding of a ligand to a protein requires the assignment of both symmetric and antisymmetric vibrational modes. The symmetric modes of isotopically enriched enzyme-bound ligands can be obtained by Raman difference spectroscopy, but until now, the antisymmetric modes, which require IR difference spectroscopy, have not been generally accessible. We have developed the methodology needed to perform IR difference spectroscopy, assign the antisymmetric modes, and accurately describe bonding. The method is used to assess the bonding changes that occur as Mg.GDP and Mg.GTP move from solution into the active site of RAS. Binding to RAS opens the nonbridging, O--P--O angle of the gamma-phosphate of GTP by 2.7 degrees, yet the angular freedom (dispersion of the O--P--O angle) of the gamma-phosphate is comparable to that in solution. In contrast, the motion of the beta-phosphate of GDP is highly restricted, suggesting that it positions the gamma-phosphate for nucleophilic attack. The beta,gamma-bridging O-P bond of bound GTP is slightly weakened, being lengthened by 0.005 A in the active site, corresponding to a bond order decrease of 0.012 valence unit (vu). The observed binding changes are consistent with a RAS-mediated hydrolysis mechanism that parallels that for solution hydrolysis.
Cheng
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Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Sukal
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S
Deng
H
H
Leyh
T S
TS
Callender
R
R
D004578:Electron Spin Resonance Spectroscopy; D006153:Guanosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D010100:Oxygen / Q000378:metabolism; D010103:Oxygen Isotopes / Q000378:metabolism; D011485:Protein Binding; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013379:Substrate Specificity
D008024:Ligands; D010103:Oxygen Isotopes; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D010100:Oxygen