Abstract
A fluorescent derivative of ATP, ε-ATP, was used to adenylylate glutamine synthetase (EC 6.3.1.2) from Escherichia coli enzymatically. The ε-adenylylated enzyme exhibits similar catalytic properties and inhibitor susceptibility to those of the naturally adenylylated enzyme. The fluorescence properties of the ε-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 Mn2+ to the ε-adenylylated enzyme is accompanied by a decrease of ε-adenosine fluorescence as compared to the effect observed for the Mg2+ 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 ε-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 Mn2+-dependent glutamine synthetase causes enhancement of the ε-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 Mg2+ and Mn2+ stabilize different conformational states of the enzyme.
Keywords: ε-ATP, ligand-induced conformational changes
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