Oxidative modification of Escherichia coli glutamine synthetase. Decreases in the thermodynamic stability of protein structure and specific changes in the active site conformation.

M T Fisher,E R Stadtman

doi:10.1016/s0021-9258(18)46028-0

Abstract

Metal catalyzed oxidation of specific amino acid residues has been proposed to be an important physiological mechanism of marking proteins for proteolytic degradation. After initial oxidative inactivation of dodecameric Escherichia coli glutamine synthetase (GS), the integrity of the GS active site and protein structure was assessed by monitoring ATP binding, observing a susceptibility of GS to tryptic cleavage, and comparative thermodynamic analysis. The tryptic cleavage rates of an active site linked central loop were significantly accelerated for the oxidized conformer. This tryptic cleavage was essentially prevented in the presence of glutamate for native GS but not for the oxidized conformer. The integrity of the ATP binding site in the oxidized GS was substantially altered as indicated by the reduction in fluorescence enhancement associated with ATP binding. Decreases in the free energies of quaternary protein structure and subunit interactions due to oxidative modification were determined by temperature and urea induced unfolding equilibrium measurements. Comparative thermal stability measurements of a partial unfolding transition indicated that the loss in stabilization free energy for the oxidized GS conformer was 1.3 kcal/mol dodecamer. Under alkaline conditions, the urea-induced disruption of quaternary and tertiary structures of oxidized and native GS were examined. This comparative analysis revealed that the free energies of the subunit interactions and unfolding of the dissociated monomers for oxidized GS were decreased by 1.5 and 1.7 kcal/mol, respectively. Our results suggest that small free energy decreases in GS protein structural stability of only 1-2 kcal/mol may be responsible for the selective proteolytic turnover of the oxidized GS.

Highlights

From the Section on Enzymes, Laboratoryof Biochemistry, Nationul Heart, Lung, andBlood Institute, National Institutesof Health, Bethesda, Maryhnd20892
Iron-catalyzed oxidation of amino acid residues located at the metal ion binding sites of E. coli glutamine synthetase has been proposed to be one mechanism by whichthe enzyme is marked for proteolytic clearance
Results presented demonstrate that this localized modification alters the binding characteristics of the enzyme for glutamate and ATP,e.g. substrate-induced conformational changes, those characterized by ATP-and glutamate-induced tryptophan fluorescence enhancement andcentral loop movements, are decreased or disappeared

Summary

Oxidative Modification oEf scherichia coli Glutamine Synthetase

DECREASES IN THE THERMODYNAMIC STABILITY OF PROTEINSTRUCTURE AND SPECIFIC CHANGES IN THE ACTIVE SITE CONFORMATION*. Localized damage of quaternary protein structure and subunit interac- can occur when activated oxygen species such as hydroxyl tions due to oxidative modification were determined radicals are formed at putative metalbinding sites in proteins by temperature and urea induced unfolding equilib- [2].The metals iron ocropper can replace the intrinsic metals rium measurements Comparative thermalstability such as Mg+,Mn2+, Ca", ZnZ+,or are themselves natural measurements of a partial unfolding transition indi- metal cofactors. Oxygen will react preferentially with amino acid residues in the vicinity of the metal binding site Both global and localized oxidative damage to proteins results in their increased susceptibility to proteolytic attack [6, 7].

MATERIALS AND METHODS

RESULTS

Thermodynamic Stability and Active Site Changes in Oxidized GS

DISCUSSION