Calorimetric and equilibrium binding studies of the interaction of substrates with glutamine synthetase of Escherichia coli

Abstract

Interactions of two substrates (L-glutamine and ADP) and Mn2+ ions with glutamine synthetase from Escherichia coli have been studied by calorimetry and equilibrium dialysis techniques. In addition, the use of calorimetry for establishing separateness of binding sites of different enzyme ligands is considered. The thermodynamic parameters for the sequential and simultaneous binding of L-glutamine and ADP to the unadenylylated Mn-enzyme have been determined. Thermal saturation curves for the binding of L-glutamine to the enzyme in the presence and absence of ADP were obtained at pH 7.1 and 30 °C. With saturating ADP and without ADP present. AG' values for L-glutamine binding are -3.83 and 16, NO.

Thermodynamics of active-site ligand binding to Escherichia coli glutamine synthetase

“…The binding of either L-Met-(S')-sulfoximine or L-glutamate to the Mn-enzyme complex was less exothermic in the presence than in the absence of AMP-PNP (Figure 6). Previously, ADP was found to make the binding of L-glutamine ~24% less exothermic while increasing 4.3-fold the affinity of the Mn-enzyme for L-glutamine (Shrake et al, 1977). Also, enthalpy changes for binding L-glutamate to the ADP-Mg-enzyme and ADP-P¡-Mg-enzyme complexes were -7.7 and -4.1 kcal/mol, respectively, at pH 7.2 and 303 K, although in this case the binding of ADP + P¡ to active sites substantially decreased the affinity for L-glutamate (Shrake et al, 1978).…”

“…In contrast, the binding of L-glutamate is less exothermic and the apparent ordering effect (as judged by the negative AS value) is less, possibly due to charge neutralization and changes in hydration waters. The binding of ADP is exothermic as is the binding of P¡ to the ADP-Mg-enzyme complex (-1.1 kcal/mol; Shrake et al, 1977Shrake et al, , 1978. In contrast, the binding of ATP or AMP-PNP is endothermic.…”

“…IN^icrocalorimetry has been used to obtain reliable thermodynamic information on protein-ligand interactions (Barisas & Gill, 1978; Biltonen & Langerman, 1979;Ross & Subramanian, 1981; Hinz, 1983;Chen & Wadso, 1982). For Escherichia coli glutamine synthetase, calorimetry has been used to assess proton effects in metal ion and substrate binding and to establish separateness of binding sites for feedback inhibitors and substrates (Hunt et al, 1972;Shrake et al, 1977Shrake et al, , 1978Ross & Ginsburg, 1969; Gorman & Ginsburg, 1982). This paper presents thermodynamic parameters for the interactions of glutamine synthetase with active-site ligands obtained by using the nonhydrolyzable ATP analogue AMP-PNP,1 lglutamate, L-Met-(S)-sulfoximine (an analogue of lglutamate), and the transition-state analogue L-Met-(-S)sulfoximine phosphate.…”

Distance determinations between the metal ion sites of Escherichia coli glutamine synthetase by electron paramagnetic resonance using chromium(III)-nucleotides as paramagnetic substrate analogs

“…A distance of 7.1 Á between Mn and Cr was obtained by analysis of these EPR data using (jlutamine synthetase from Escherichia coli is composed of 12 identical subunits and is known to catalyze several reactions in addition to the biosynthesis of glutamine from glutamate, ammonia, and ATP (Stadtman & Ginsburg, 1974). The enzyme shows an absolute requirement for two divalent cations per subunit for catalysis to occur (Hunt et al, 1975).The unadenylylated enzyme binds Mn(II) at these two sites with affinities that differ by approximately two orders of magnitude in the absence of substrates (Denton & Ginsburg, 1969;Hunt et al, 1975;Villafranca et al, 1976;Shrake et al, 1977). The first ("tight") metal ion site is known to produce conformational changes in the protein but may also be near the catalytic site (Villafranca et al, 1976).…”

“…The unadenylylated enzyme binds Mn(II) at these two sites with affinities that differ by approximately two orders of magnitude in the absence of substrates (Denton & Ginsburg, 1969;Hunt et al, 1975;Villafranca et al, 1976;Shrake et al, 1977). The first ("tight") metal ion site is known to produce conformational changes in the protein but may also be near the catalytic site (Villafranca et al, 1976).…”