Role of Mn²⁺ and Mg²⁺ in catalysis and regulation of Aspergillus niger glutamine synthetase

Abstract

The kinetic data On the effect of Mn²⁺, Mg²⁺ .and ATP on the Aspergillus niger glutamine synthetase activity analysed according to the isovelocity method [London W P & Steck T L (1969) Biochemistry; 8, 1767J revealed that the enzyme under physiological conditions is probably an Mn(II) enzyme. Excess of ATP or Mn²⁺ beyond the concentrations required to form the metal ion-nudeotide compiex inhibited both the Mg²⁺ and Mn²⁺ supported glutamine synthetase activity, whereas excess Mg²⁺ was not inhibitory. These results along with the inhibuion of enzyme activity by Ca²⁺, Zn²⁺, Co²⁺. GTP and EDTA indicated that the interactions of the enzyme with Mn²⁺and Mg²⁺ were different. Supporting evidence for the proposed kinetic mechanism was obtained by the protection afforded by low concentrations of Mn²⁺(< 100 µM) against inactivalion of the enzyme by N-ethylmaleimide or phenylglyoxal. However, ATP enhanced the rates of inactivation by both modifying agents K_Mn²⁺ values of 52 and 14 µM calculated from the protection afforded by this metal ion against inactivation by phenylglyoxal and N-ethylmaleimide suggested the presence of a high affinity Mn²⁺ binding site on the enzyme, in addition to the binding of Mn²⁺ ATP complex at the active site. These results permit the conclusion that A.niger glutamine synthetase may be an Mn(1I) dependent enzyme under physiological conditions and the metal ion in addilion to serving as a substrate when complexed with A TP may have an additional role in protecting the enzyme against inactivation.