Kinetic Properties and Ammonium-dependent Regulation of Cytosolic Isoenzymes of Glutamine Synthetase in Arabidopsis

Keiki Ishiyama, Eri Inoue, Akiko Watanabe-Takahashi, Mitsuhiro Obara, Tomoyuki Yamaya, Hideki Takahashi

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148 Citations (Scopus)

Abstract

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, cytosolic GS (GS1) was accumulated in roots when plants were excessively supplied with ammonium; however, the GS activity was controlled at a constant level. The discrepancy between the protein content and enzyme activity of GS1 was attributable to the kinetic properties and expression of four distinct isoenzymes encoded by GLN1;1, GLN1;2, GLN1;3 and GLN1;4, genes that function complementary to each other in Arabidopsis roots. GLN1;2 was the only isoenzyme significantly up-regulated by ammonium, which correlated with the rapid increase in total GS1 protein. GLN1;2 was localized in the vasculature and exhibited low affinities to ammonium (Km = 2450 ± 150 μM) and glutamate (Km = 3.8 ± 0.2 mM). The expression of the counterpart vascular tissue-localizing low affinity isoenzyme, GLN1;3, was not stimulated by ammonium; however, the enzyme activity of GLN1;3 was significantly inhibited by a high concentration of glutamate. By contrast, the high affinity isoenzyme, GLN1;1 (Km for ammonium < 10 μM; Km for glutamate = 1.1 ± 0.4 mM) was abundantly accumulated in the surface layers of roots during nitrogen limitation and was down-regulated by ammonium excess. GLN1;4 was another high affinity-type GS1 expressed in nitrogen-starved plants but was 10-fold less abundant than GLN1;1. These results suggested that dynamic regulations of high and low affinity GS1 isoenzymes at the levels of mRNA and enzyme activities are dependent on nitrogen availabilities and may contribute to the homeostatic control of glutamine synthesis in Arabidopsis roots.

Original languageEnglish
Pages (from-to)16598-16605
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number16
DOIs
Publication statusPublished - 2004 Apr 16

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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