Purification and characterization of glutamine synthetase from the unicellular cyanobacterium Synechococcus RF-1

Abstract

Glutamine synthetase (GS; EC 6.3.1.2) from the Synechococcus RF-1 was purified to homogeneity by ion exchange, molecular sieving, and hydroxyapatite chromatographies. The native enzyme has a molecular mass of about 456kDa, and the molecular mass of its subunit was about 56kDa. Electron micrographs of the enzyme revealed two parallel protein layers in cubic symmetry with quaternary structure. The actual data indicated that the enzyme could consist of eight identical subunits. The enzyme had an apparent Km value for L-glutamate of 233mM, but it exhibited positive cooperativity for ATP (n(subscript H)=1.5 and S0.5=0.94 mM) and NH4C1(n(subscript H)=2, and S0.5=1.33 mM) in the biosynthetic assay. The enzyme had apparent Km values for L-glutamine and hydroxylamine of 8.70 mM and 7.04 mM, respectively, in the transferase assay. This enzyme was quite stable in Tris-HCI buffer (pH 7.5) containing EDTA, MgCl2, and 2-mercaptoethanol. The pH optima for both the biosynthetic and transferase activities of the enzyme were 8.1 and 8.4, respectively. The enzyme required a divalent metal ion as an activator. Mg(superscript 2+) was the most effective metal ion for biosynthetic activity, followed by Co(superscript 2+). Mn(superscript 2+) was the most effective metal ion for transferase activity. Ca(superscript 2+) and Mn(superscript 2+) strongly inhibited Mg(superscript 2+)-supported biosynthetic activity, but Co(superscript 2+) stimulated it.