The physiology of stringent factor (ATP:GTP 3'-diphosphotransferase) in Escherichia coli.
The enzyme ATP:GTP 3'-diphosphotransferase catalyzes the transfer of the beta, gamma-pyrophosphate of ATP to the 3' position of GTP or GDP. The amounts of enzyme were measured in cell extracts of a relA+ strain of E. coli grown at different growth rates between 0.4 and 1.9 generations per hour, using precipitation with specific antibodies to purify the enzyme. The amount of enzyme was found to be a constant fraction of total protein at all growth rates corresponding to about 45 molecules of enzyme per genome equivalent of DNA. The purified enzyme has little catalytic activity by itself but has to be activated either by a complex of 70S ribosomes, mRNA and uncharged tRNA or by a solvent like ethanol at a concentration of about 20%. The kinetic constants of the enzyme for the transfer pyrophosphate from ATP to GTP in the ribosome-activated state were determined. The Vmax was estimated to be 140 mumol/min X mg at 37 degrees C and the S0.5 values for GTP and ATP were 0.35 and 0.53 mM, respectively. The reaction was estimated to have an equilibrium constant of about 300. In the pyrophosphate transfer from ATP to GDP the Vmax was estimated to be 90 mumol/min X mg at 37 degrees C and the S0.5 for GDP as 0.3 mM. During amino acid starvation of a relA+ strain of E. coli the amounts of enzyme and the catalytic capacity of the enzyme are sufficient to maintain the observed ppGpp levels in the cells at all growth rates.
D000255:Adenosine Triphosphate / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D001277:GTP Pyrophosphokinase / Q000037:antagonists & inhibitors / Q000502:physiology*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007163:Immunosorbent Techniques; D007700:Kinetics; D010770:Phosphotransferases / Q000502:physiology*; D013379:Substrate Specificity
D011756:Diphosphates; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D001277:GTP Pyrophosphokinase