Thermodynamic effects of active-site ligands on the reversible, partial unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric studies.


Abstract

Dodecameric glutamine synthetase (GS) from Escherichia coli undergoes reversible, thermally induced partial unfolding without subunit dissociation. A single endotherm for Mn.GS (+/- active-site ligands) in the presence of 1 mM free Mn2+ and 100 mM KCl at pH 7 is observed by differential scanning calorimetry (DSC). Previous deconvolutions of DSC data for Mn.GS showed only two two-state transitions (with similar tm values; 51.6 +/- 2 degrees C), and indicated that cooperative interactions link partial unfolding reactions of all subunits within the Mn.enzyme dodecamer [Ginsburg, A., & Zolkiewski, M. (1991) Biochemistry 30, 9421]. A net uptake of 8.0 equiv of H+ by Mn.GS occurs during partial unfolding, as determined in the present DSC experiments conducted with four buffers having different heats of protonation at 50 degrees C. These data gave a value of 176 +/- 12 kcal (mol of dodecamer)-1 for delta Hcal corrected for buffer protonation. L-Glutamine and L-Met-(SR)-sulfoximine stabilize the Mn.GS dodecamer through the free energies of ligand binding, and these were shown to be partially and totally released, respectively, from the 12 active sites at high temperature. Ligand effects on Tm values from DSC were similar to those from spectral measurements of Trp and Tyr exposures in two subunit domains. Effects of varying [ADP] on DSC profiles of Mn.GS were complex; Tm is increased by low [ADP] and decreased by > 100 microM free ADP. This is due to the exposure of an additional low-affinity ADP binding site per GS subunit at high temperature with log K1' = 4.3 and log K2' = 3.6 at 60 degrees C relative to log K' = 5.5 for ADP binding at 30 degrees C, as determined by isothermal calorimetric and fluorescence titrations. Moreover, delta Hcal at > 27% saturation with ADP (corrected for ADP binding/dissociation) is approximately 80-100 kcal/mol more than in the absence of ligands. Changes in domain interactions could result from ADP bridging subunit contacts in the dodecamer. Each of the active-site ligands investigated here produces different effects on DSC profiles without uncoupling the extremely cooperative, partial unfolding reactions in the Mn.GS dodecamer. Study holds ProTherm entries: 4790 Extra Details: subunit dissociation; two-state transitions; partial unfolding;,low-affinity ADP binding; active-site ligands; cooperative

Submission Details

ID: 7juCBvXp

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Zolkiewski M;Ginsburg A,Biochemistry (1992) Thermodynamic effects of active-site ligands on the reversible, partial unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric studies. PMID:1360813
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