Affinity labeling of Escherichia coli glutamine synthetase by β, γ―Cr(III)(H2O)4ATP

The interaction of Escherichia coli glutamine synthetase with beta, gamma-Cr(III)(H2O)4ATP (CrATP) has been studied. This substitution inert nucleotide functioned as an active site directed irreversible inhibitor of glutamine synthetase in solutions containing 15 mM MgCl2, 100 mM KCl, and 10 mM Pipes (pH 6.6). The inactivation reaction followed pseudo-first order saturation kinetics which demonstrated reversible binding of CrATP prior to the formation of inactive enzyme. CrATP was shown to be a competitive inhibitor versus MgATP. Also, significant protection was afforded by MgATP indicating that CrATP inactivates at the active site. Partial protection was afforded by glutamate or inorganic phosphate while inactivation was enhanced by Mn(II). The stoichiometry of CrATP incorporation was approximately one molecule per enzyme subunit, determined spectrophotometrically. Both the delta and lambda isomers of CrATP bound to glutamine synthetase, but only the lambda isomer was an active site directed irreversible inhibitor.