Interaction of substrates with glutamine synthetase after limited proteolysis.
Previous studies [Dautry-Varsat, A., Cohen, G. N., & Stadtman, E.R. (1979) J. Biol. Chem. 254, 3124-3128; Lei, M., Aebi, U., Heidner, E. G., & Eisenberg, D. (1979) J. Biol. Chem. 254, 3129-3134] have shown that Escherichia coli glutamine synthetase (GS) can be cleaved by proteases to form a limited digestion species called nicked glutamine synthetase (GS). The present study gives the amino acid sequence of the protease-sensitive region of glutamine synthetase. The present study also shows that GS is enzymatically active, but this activity is low compared to the activity of GS. The apparent Michaelis constant value for glutamate was 90 mM for GS as compared to 3 mM for GS, while the Michaelis constant values for ATP were similar for GS and GS*. The dissociation constant values for ATP, as determined by intrinsic fluorescence measurements, were similar for GS and GS*. Glutamate decreased the dissociation constant value of ATP for GS because of synergism between the two binding sites; glutamate did not decrease the dissociation constant value of ATP for GS*. The glutamate analogue methionine sulfoximine bound very tightly to GS and inactivated the enzyme in the presence of ATP. Methionine sulfoximine did not appear to bind to GS* and did not inactivate GS* in the presence of ATP. The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine bound to GS and inactivated the enzyme by forming a covalent bond with it. Glutamate accelerated this inactivation because of the synergism between the ATP and glutamate binding sites of GS.(ABSTRACT TRUNCATED AT 250 WORDS)
D001665:Binding Sites; D001667:Binding, Competitive; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008717:Methionine Sulfoximine / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D010446:Peptide Fragments; D008717:Methionine Sulfoximine; D010450:Endopeptidases; D005974:Glutamate-Ammonia Ligase