Studies onAspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grownAspergillus niger was increased 3–5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH ₄ ⁺ , and further, the enzyme is repressed by increasing concentrations of NH ₄ ⁺ . In contrast to other micro-organisms, theAspergillus niger enzyme was neither specifically inactivated by NH ₄ ⁺ or L-glutamine nor regulated by covalent modification. Glutamine synthetase fromAspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn²⁺ or Mg²⁺-dependent synthetase and Mn²⁺-dependent transferase activity. Aspergillusniger glutamine synthetase was completely inactivated by two mol of phenyl-glyoxal and one mol of N-ethylmaleimide with second order rate constants of 3.8 M^-1 min^-1 and 760 M^-1 min^-1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH ₄ ⁺ , Mn²⁺ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.