Purification and properties of glutamine synthetase from the archaebacterium Halobacterium salinarium

Abstract

Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the halophilic archaebacterium Halobacterium salinarium. The enzyme was purified 300-fold to homogeneity with 30% yield. By gel filtration and SDS gel electrophoresis, it was shown that the enzyme has a native molecular weight of 495,000 and a subunit molecular weight of 62,000. This indicates an octameric quaternary structure. The amino acid composition and the isoelectric point of 4.9 are similar to other GSs. The enzyme shows highest stability in 4 M NaCl or KCl and at temperatures up to 45°C. Lower salt concentrations or higher temperatures lead to rapid and irreversible denaturation. By low concentrations of Mg2+ or Mn2+, the salt dependence was decreased and the thermostability increased. Mg2+ or Mn2+ are essential cofactors. The two resulting activities show differences in pH and salt concentrations required for optimal activity, different Km-values and different sensitivity to inhibition by amino acids. The enzyme is not adenylylated like the GS from some eubacteria but cytidylylated. The covalently bound CMP increases Mn2+-and Mg2+-dependent activities at a different extent.