Fluorescence Studies of Glutamine Synthetase from Escherichia coli: ϵ-Adenylylated and Unadenylylated Enzymes

Abstract

A fluorescent derivative of ATP, ϵ -ATP, was used to adenylylate glutamine synthetase from E. 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 of unadenylylated subunits; and at high ADP concentration, ADP causes not only enhancement of the tryptophan 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.

Studies of ligand-induced fluorescence changes of the unadenylylated enzyme were carried out using ADP, P_i and arsenate. Enhancement of the tryptophan fluorescence is obtained with the addition of each of these ligands to the unadenylylated enzyme in the presence of either Mg²+ or Mn²⁺. The absolute magnitude of total increase in fluorescence intensity is considerably greater for the Mn²+ enzyme. It is demonstrated that the fluorescence changes noted upon successive saturation of the enzyme by the addition of ADP followed by P_i or P_i followed by ADP corresponds to random addition of these ligands to the enzyme. A similar result is obtained for the ADP — As_i system. Despite this random addition mechanism, it is significant to note that the binding of ADP to the enzyme is enhanced in the presence of P_i (or As_i), Similarly the P_i (or As_i) binding is higher in the presence of ADP. This enhanced binding is considerably more pronounced for the Mn²⁺ enzyme than for Mg²⁺ enzyme.

Department of Chemistry, The Catholic University of America, Washington, D. C. 20017. Recipient of the National Heart and Lung Institute Special Research Fellowship Award.