Interaction of substrates with glutamine synthetase after limited proteolysis

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Abstract
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 Km value for glutamate was 90 mM for GS* as compared to 3 mM for GS, while the Km 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 2 binding sites; glutamate did not decrease the dissociation constant value of ATP for GS*. The glutamate analog 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 analog 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. 5''-[p-(Fluorosulfonyl)benzoyl]adenosine bound to GS* at least as tightly as to GS, but inactivated GS* 10-fold more slowly than GS. Glutamate did not increase the rate of inactivation of GS* by 5''-[p-(fluorosulfonyl)benzoyl]adenosine. The lower enzymatic activity of GS* compared to GS is due to an apparent decrease in the binding affinity of GS* for glutamate, and to a loss of the synergism between ATP and glutamate binding.