Mechanism of the ribosome-dependent uncoupled GTPase reaction catalyzed by polypeptide chain elongation factor G.
At low NH4-+ concentrations, 50S ribosomal subunits from E. coli were fully active in the absence of 30S ribosomal subunits, in forming a complex with the polypeptide chain elongation factor G (EF-G) and guanine nucleotide (ternary complex formation), and also in supporting EF-G dependent hydrolysis of GTP (uncoupled GTPase reaction).  However, both activities were markedly inhibited on increasing the concentration of the monovalent cation, and at 160 mM NH4-+, the optimal concentration for polypeptide synthesis in a cell-free system, almost no activity was observed with 50S ribosomes alone.  It was found that the inhibitory effect of NH4-+ was reversed by addition of 30S subunits.  Thus, at 160 mM NH4-+, only 70S ribosomes were active in supporting the above two EF-G dependent reactions, whereas at 20 mM NH4-+, 50S ribosomes were almost as active as 70S ribosomes.  Kinetic studies on inhibition by NH4-+ of the formation of 50S ribosome-EF-G-guanine nucleotide complex, indicated that the inhibition was due to reduction in the number of active 50S ribosomes which were capable of interacting with EF-G and GTP at higher concentrations of NH4-+.  The inhibitory effects of NH4-+ on ternary complex formation and the uncoupled GTPase reaction were markedly influenced by temperature, and were much greater at 0 degrees than at 30 degrees.  A conformational change of 50S subunits through association with 30S subunits is suggested.
D000641:Ammonia / Q000494:pharmacology*; D002499:Centrifugation, Density Gradient; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010445:Peptide Elongation Factors; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D013696:Temperature; D013997:Time Factors
D010445:Peptide Elongation Factors; D000641:Ammonia; D010744:Phosphoric Monoester Hydrolases; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D008274:Magnesium