Nucleotide binding and GTP hydrolysis by the 21-kDa product of the c-H-ras gene as monitored by proton-NMR spectroscopy.
Proton-NMR signals in the downfield region (below approximately 10 ppm) have been shown to provide a useful spectroscopic window to monitor the binding of guanine nucleotides to the active site of GTP/GDP-binding proteins via H-bonds, as specified here by the 21-kDa product of the c-H-ras gene (p21). The time course of the intensity change of certain peaks upon addition of GTP to nucleotide-free p21 corresponds to the GTP hydrolysis rate as determined by HPLC. Though there are fewer potential H-bond acceptors in the GDP-bound protein than in the GTP complex, more downfield peaks are found in the former complex, suggesting tighter binding of GDP. Moreover, inspection of the downfield proton-NMR spectra permits rapid detection of subtle changes of the active site induced by complexation with slowly hydrolyzing GTP analogues resulting from mutations of the amino acid sequence, especially in the phosphate binding loop. Our studies strongly suggest that no major conformational change of the phosphate-binding region occurs upon nucleotide complexation that precedes the catalytic step. Besides, it is suspected that the Ser17 hydroxyl group is involved in nucleotide binding and GTP hydrolysis.
Löw
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A
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Sprinzl
M
M
Limmer
S
S
D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D011905:Genes, ras; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008970:Molecular Weight; D011487:Protein Conformation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011522:Protons; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras)