Reactivation of glutamine synthetase from Escherichia coli after auto-inactivation with L-methionine-S-sulfoximine, ATP, and Mn2+.
Escherichia coli glutamine synthetase auto-inactivated with L-methionine-S-sulfoximine and ATP can be completely reactivated at pH 3.5-4.6 in 1 M KCl and 0.4 M (NH4)2SO4. Both unadenylylated and adenylylated magnesium and manganese enzymes can be reactivated. Reactivation of fully inactivated enzyme is first order (t1/2 approximately 2 min at pH 4.1 and 37 degrees C) and coincides with the stoichiometric release of 0.95 +/- 0.05 eq each of L-methionine-S-sulfoximine phosphate and ADP and 2.0 +/- 0.2 eq of Mn2+ from each subunit. The rate of reactivation increases with decreasing pH and is proportional to the 3rd to 4th power of the hydrogen ion activity; the protonation of 3-4 carboxylic acid groups/subunit therefore may be required to disrupt the enzyme complex. Reactivation rate also increases with increasing KCl concentrations and temperature, with an Arrhenius activation energy of approximately 26 Kcal/mol, suggesting that some protein structural perturbation is required to disrupt the complex. Upon neutralization os reactivation solutions, the ligands and metal ions recombine with the enzyme resulting in its complete reinactivation. Thus, inactivation of glutamine synthetase at neutral pH is due to the extremely tight, but reversible, binding of L-methionine-S-sulfoximine phosphate, ADP, and Mn2+ (K'A greater than 10(12) M-1 for ADP) to the enzyme. Under certain conditions, ATP or ADP can partially inhibit inactivation. Excess ADP inhibits reinactivation by L-methionine-S-sulfoximine phosphate, and ATP has a complex effect on the rate and extent of the autoinactivation reaction.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology*; D008717:Methionine Sulfoximine / Q000494:pharmacology*; D009994:Osmolar Concentration; D013816:Thermodynamics
D008717:Methionine Sulfoximine; D008345:Manganese; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase