Epsilon-adenylylated glutamine synthetase: an internal fluorescence probe for enzyme conformation.
A fluorescent derivative of ATP, epsilon-ATP, was used to adenylylate glutamine synthetase (EC 6.3.1.2) from Escherichia coli enzymatically. The epsilon-adenylylated enzyme exhibits similar catalytic properties and inhibitor susceptibility to those of the naturally adenylylated enzyme. The fluorescence properties of the epsilon-adenosine and of tryptophan residues of the enzyme were used to study ligand-induced conformational changes involving alterations of the tryptophan regions and the adenylylation site of the protein. Binding of Mn(2+) to the epsilon-adenylylated enzyme is accompanied by a decrease of epsilon-adenosine fluorescence as compared to the effect observed for the Mg(2+) binding. An ADP binding study shows that at low ADP concentration, ADP causes enhancement of the tryptophan fluorescence only, reflecting the binding to unadenylylated subunits; and at high ADP concentration, ADP causes not only enhancement of the fluorescence, but also a quenching of the fluorescence of enzyme-bound epsilon-AMP, reflecting binding to the adenylylated subunits. Dissociation constants calculated from these fluorescence changes agree well with those determined from binding studies of ADP to adenylylated and unadenylylated enzymes. Binding of the feedback inhibitor, alanine, to Mn(2+)-dependent glutamine synthetase causes enhancement of the epsilon-AMP fluorescence, from which a dissociation constant of 1.5 mM was calculated for the inhibitor. The fluorescence changes observed due to ligands binding suggest that Mg(2+) and Mn(2+) stabilize different conformational states of the enzyme.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008274:Magnesium; D008345:Manganese; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000249:Adenosine Monophosphate; D008345:Manganese; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium