Aggregation and thermo-inactivation of glutamine synthetase from an extreme thermophile, Bacillus caldolyticus.

The extreme thermophile, Bacillus caldolyticus, contains two regulatory isoforms of glutamine synthetase (glutamate-ammonia ligase, EC 6.3.1.2), E-I and E-II, produced as separate gene products. Light scattering and electron microscopy data indicate that these thermophilic enzymes aggregate to higher molecular weight species in two stages: initial polymerization of native dodecamers, followed by 'melting' of the aggregated species to produce amorphous denatured protein. The initial stages of the aggregation occurred at temperatures below those for time-dependent denaturation, especially for E-II. In contrast, mesophilic (B. subtilis) enzyme showed no evidence of temperature-dependent aggregation. Thus, aggregation may be a stabilizing mechanism for the thermophilic systems. Bound metal ions and substrates caused dramatic increases in the temperatures at which aggregation and loss of activity occurred for thermophilic enzymes. Certain combinations of ligands (e.g., MnATP + L-glutamate) acted synergistically, so that these complexes denatured only above 90 degrees C. Various models were considered for heat-driven aggregation followed by denaturation, plus ligand stabilization. Taken together, the data are most consistent with unfolding of subunits within the dodecameric unit, rather than unfolding to monomers prior to aggregation.