UREA-INDUCED DISSOCIATION AND UNFOLDING OF DODECAMERIC GLUTAMINE-SYNTHETASE FROM ESCHERICHIA-COLI - CALORIMETRIC AND SPECTRAL STUDIES

Urea-induced dissociation and unfolding of manganese glutamine synthet ase (Mn . GS) have been studied at 37 degrees C (pH 7) by spectroscopi c and calorimetric methods. In 0 to similar to 2 M urea, Mn . GS retai ns its dodecameric structure and full catalytic activity. Mn . GS is d issociated into subunits in 6 M urea, as evidenced by a 12-fold decrea se in 90 degrees light scattering and a monomer molecular weight of 51 ,800 in sedimentation equilibrium studies. The light scattering decrea se in 4 M urea parallels the time course of Trp exposure but occurs mo re rapidly than changes in secondary structure and Tyr exposure. Early and late kinetic steps appear to involve predominantly disruption of intra-ring and inter-ring subunit contacts, respectively, in the layer ed hexagonal structure of Mn . GS. The enthalpies for transferring Mn . GS into urea solutions have been measured by titration calorimetry. After correcting for the enthalpy of binding urea to the protein, the enthalpy of dissociation and unfolding of Mn . GS is 14 +/- 3 cal/g. A net proton uptake of similar to 50 H+/dodecamer accompanies unfolding reactions. The calorimetric data are consistent with urea binding to multiple, independent sites in Mn . GS and the number of binding sites increasing similar to 9-fold during the protein unfolding.