Rapid transfer of oxygens from inorganic phosphate to glutamine catalyzed by Escherichia coli glutamine synthetase.
Measurements are reported on certain isotopic fluxes during the net conversion of glutamine, ADP and Pi to glutamate, NH3, and ATP by Escherichia coli glutamine synthetase (adenylylated form, Mn2+ activated) in presence of a hexokinase/glucose trap to remove the ATP formed during the reaction. The results show that the transfer of oxygens from Pi to glutamine is the most rapid of the measured isotopic interchanges, over five oxygens from Pi being transferred to glutamine for each glutamate formed by net reaction. Under similar conditions, the oxygen transfer from Pi to glutamate, was stimulated somewhat by an increase in the glutamate concentration but inhibited by an increase in the ammonia concentration. The enzyme from brain or peas did not show the rapid transfer of 18O from Pi to glutamine shown by the E. coli enzyme. Deductions are also made from the data about the availability of the oxygens of gamma-carboxyl of bound glutamate for reaction. The most logical explanation of the results with the E. coli enzyme is that the gamma-carboxyl group of bound glutamate has sufficient rotational freedom so that under conditions of rapid substrate interconversion either carboxylate oxygen can participate in the reaction. The results with the pea enzyme are consistent with hindered rotation of the gamma-care additional findings make likely a relative order of certain catalytic steps for the E. coli enzyme as follows: ATP release less than NH3 release less than glutamate release less than substrate interconversion less than glutamine release and Pi release and glutamate release less than ADP release.
D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D005971:Glutamates / Q000494:pharmacology; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D012756:Sheep; D013045:Species Specificity
D005971:Glutamates; D010710:Phosphates; D005973:Glutamine; D000641:Ammonia; D005974:Glutamate-Ammonia Ligase