Some regulative properties of glutamine synthetase in cucumber leaves

Glutamine synthetase (L-glutamate: ammonia ligase (ADP) EC 6. 3. t 2) was prepared from cucumber leaves grown on ammonium medium and some regulative properties were investigated. The apparent Michaelis constant (Km) values of the various substrates and cofactors were determined. Lineweaver-Burk plots gave a Km of 4.5mM with L-glutamate, 0.74mM with ATP and 3.1 mM with NH₂OH. Metals, including Cu²⁺, Hg²⁺, Zn²⁺, Ni²⁺, Fe²⁺, and Fe³⁺, strongly inhibited the enzyme activity, Cd²⁺ did not inhibit the enzyme activity markedly, in contrast with its effect on the rice enzyme. Ca²⁺ was quite inhibitive to enzyme reaction and more than 50% of the activity was lost at 3 mM. The amino acids tested generally had no effect on the enzyme activity except alanine, which showed little but clear inhibition. Isocitrate and α-ketoglutarate were slightly promotive to enzyme activity while pyruvate and glyoxylate (24 mM) significantly inhibited the enzyme activity. Glucose-l- or -6-phosphate and fructose-6-phosphate were inhibitive to similar degrees, about 20% at 22.5 mM, and 3-phosphoglycerate (22.5 mM) markedly inhibited the enzyme activity up to 56%. Among the nucleotides tested, UTP, CTP, and GTP inhibited slightly, and marked inhibitions of H and 78% were observed after the addition of AMP (5 mM) and ADP (5 mM) respectively. Pyridoxal-5′-phosphate, which is a characteristic incompetitive inhibitor of L-glutamate and NH₂-OH, here inhibited enzyme activity significantly. On the other hand, pyridoxamine-5′-phosphate had almost no effect. The inhibition caused by the former was not recovered by the latter. This response of glutamine synthetase to both compounds was in agreement with the idea that the nitrogen status of the plant could be reflected by the ratio pyridoxamine phosphate/pyridoxal phosphate. Also this suggested that the regulative properties of glutamine synthetase with respect to pyridoxal or pyridoxamine phosphate could be understood from the point of view of the economical use of nitrogen.