Glutamine synthetase of pea leaves: divalent cation effects, substrate specificity, and other properties.
Purified glutamine synthetase from pea seedlings was most active with Mg(2+) as the metal activator, but Mn(2+) and Co(2+) were 45 to 60% and 30 to 45% as effective, respectively, when assayed at the optimal pH for each cation. The Mg(2+) saturation curve was quite sigmoid, and evidence indicates that MgATP is the active ATP substance. Co(2+) also gave a sigmoidal saturation curve, but when Mn(2+) was varied only slightly sigmoidal kinetics were seen. Addition of Mn(2+), Ca(2+), or Zn(2+) at low concentrations sharply inhibited the Mg(2+) -dependent activity, partially by shifting the pH optimum. Addition of Co(2+) did not inhibit Mg(2+)-dependent activity. The nucleotide triphosphate specificity changed markedly when Co(2+) or Mn(2+) replaced Mg(2+). Using the Mg(2+)-dependent assay, the Michaelis constant (Km) for NH(4) (+) was about 1.9 x 10(-3) M. The Km for l-glutamate was directly proportional to ATP concentration and ranged from 3.5 to 12.4 mm with the ATP levels tested. The Km for MgATP also varied with the l-glutamate concentration, ranging from 0.14 mm to 0.65 mm. Ethylenediaminetetracetic acid activated the enzyme by up to 54%, while sulfhydryl reagents gave slight activation, occasionally up to 34%.
O'neal
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Department of Biology, Carleton University, Ottawa, Canada.
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