Characterization of the GTPase reaction of elongation factor Tu. Determination of the stereochemical course in the presence of antibiotic X5108.
The stereochemical course of the GTPase of elongation factor Tu from Escherichia coli has been determined by making use of the reaction dependent on antibiotic X5108 (an N-methylated derivative of kirromycin). Guanosine 5'-(gamma-thio)triphosphate stereospecifically labeled with 17O and 18O in the gamma-position was hydrolyzed in the presence of elongation factor Tu and X5108. The configuration of the product, inorganic [16O, 17O, 18O]thiophosphate was analyzed by 31P NMR after its stereospecific incorporation into adenosine 5'-(beta-thio)triphosphate. The analysis showed that the hydrolysis proceeds with inversion of configuration at the transferred phosphorus, implying that there is not a phosphoenzyme intermediate. Incubation of [beta gamma-18O, gamma-18O3]GTP with elongation factor Tu leaves the 18O-labeling unaltered, as shown by 31P NMR. No exchange of oxygens with water nor beta gamma-beta positional isotope exchange occurs, implying that not even transient cleavage can occur with the elongation factor alone. Only on interaction with X5108, kirromycin, or ribosomes does the cleavage occur, most likely by a single step, in-line transfer of the terminal phosphorus from GDP to a water oxygen. These properties of the GTP hydrolysis mechanism of elongation factor Tu are similar to those of elongation factor G.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000899:Aurodox / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism
D000900:Anti-Bacterial Agents; D010445:Peptide Elongation Factors; D013873:Thionucleotides; D000899:Aurodox; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu