Isotope-exchange enhancement studies of Escherichia coli glutamine synthetase.
Isotope-exchange enhancement studies, a variation on positional isotope-exchange enhancement as described by Raushel and Garrard [Raushel, F. M., & Garrard, L. J. (1984) Biochemistry 23, 1791-1795], are used to establish the point in the biosynthetic reaction of Escherichia coli glutamine synthetase at which gamma-glutamyl phosphate is formed. In these experiments, the behavior of the reverse biosynthetic reaction, i.e., the reaction of ADP, L-glutamine, and phosphate to form NH4+, L-glutamate, and ATP, is examined as a function of the concentration of ammonium ion. By varying the concentration of NH4+, the ratio of the velocity of isotope exchange to the velocity of net reaction, as measured by the rate of 18O depletion from labeled phosphate and the rate of production of L-glutamate, respectively, can be modulated in a mechanism-dependent manner. Evidence is presented demonstrating the presence of a branch point in the mechanism. The enzyme-ATP-glutamate complex may partition in two ways, one involving binding of ammonium ion and the other involving the chemical transformation to form the enzyme-ADP-gamma-glutamyl phosphate complex. The alternate pathways then rejoin upon formation of the enzyme-ADP-NH4+-gamma-glutamyl phosphate complex. Because of the branch point, there is no absolute requirement that ammonium ion be absent or present in order for the formation of gamma-glutamyl phosphate to occur. At high concentrations of ammonia, one pathway through the branch can be eliminated, effectively making that portion of the pathway ordered, with ATP, L-glutamate, and NH4+ binding consistent with our previously reported steady-state kinetic mechanism [Meek, T. D., & Villafranca, J. J. (1980) Biochemistry 19, 5513-5519].
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D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008433:Mathematics; D010103:Oxygen Isotopes
D010103:Oxygen Isotopes; D005974:Glutamate-Ammonia Ligase