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 almost-equal-to 10 p pm) have been shown to provide a useful spectroscopic window to monito r the binding of guanine nucleotides to the active site of GTP/GDP-bin ding 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 cer tain peaks upon addition of GTP to nucleotide-free p2l corresponds to the GTP hydrolysis rate as determined by HPLC. Thou-h there are fewer potential H-bond acceptors in the GDP-bound protein than in the GTP co mplex, more downfield peaks are found in the former complex, suggestin g tighter binding, of GDP. Moreover, inspection of the downfield proto n-NMR spectra permits rapid detection of subtle changes of the active site induced by complexation with slowly hydrolyzing GTP analogues res ulting from mutations of the amino acid sequence, especially in the ph osphate binding loop. Our studies strongly suggest that no major confo rmational change of the phosphate-binding region occurs upon nucleotid e complexation that precedes the catalytic step. Besides, it is suspec ted that the Ser17 hydroxyl group is involved in nucleotide binding an d GTP hydrolysis.