A CALORIMETRIC STUDY OF BINDING OF 2 FEEDBACK INHIBITORS TO GLUTAMINE SYNTHETASE FROM ESCHERICHIA COLI

The apparent heats of binding of two feedback inhibitors, alone or mixed, at saturating concentrations (in 0.02 m imidazole, 0.1 m KCl, and 0.001 m MnCl2, pH 7.07), to the Mn2+-activated form of the glutamine synthetase from Escherichia coli have been measured by calorimetry at 25°. Where ∆H is expressed as calories per mole of subunit of 50,000 molecular weight, ∆H = -7350 and ∆H = -2000 for ltryptophan and adenosine 5′-monophosphate binding, respectively. For a saturating mixture of L-tryptophan and adenosine 5′-monophosphate, a ∆H value of -9600 cal/mole was measured which is about equal to the sum of the enthalpies of the binding of the individual effectors to the enzyme. The additivity of the measured ∆H values demonstrates that the Mn2+ form of glutamine synthetase, when saturated with 12 equiv of each effector/mole of enzyme, has separate binding sites for L-tryptophan and adenosine 5′-monophosphate. The ability to measure reaction heats in the 0.2-5-mcal range suggests that calorimetry will be useful in studying the interaction between this enzyme and even more weakly, or strongly, bound effectors. There is no correlation between the measured ∆H values and the apparent free energies of binding of either L-tryptophan or adenosine 5′-monophosphate to glutamine synthetase in the same buffer system. Apparent entropy changes of about -11 and +11 cal deg-1 (mole of subunit)-1 were estimated for the binding of L-tryptophan and adenosine 5′-monophosphate, respectively. These entropy changes are 12 times greater for the entire glutamine synthetase molecule, and are therefore quite large. When both effectors are bound simultaneously to the enzyme, the accompanying total apparent entropy change is nearly zero. © 1969, American Chemical Society. All rights reserved.