λ-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization

Abstract

λ-Glutamylcysteine synthetase activity (EC 6.3.2.2) was analysed in Sephacryl S-200 eluents of extracts from cell suspension cultures ofNicotiana tabacum L. cv. Samsun by determination of λ-glutamylcysteine as its monobromobimane derivative. The enzyme has a relative molecular mass (Mr) of 60000 and exhibits maximal activity at pH 8 (50% at pH 7.0 and pH9.0) and an absolute requirement for Mg(2+). With 0.2mM Cd(2+) or Zn(2+), enzyme activity was reduced by 35% and 19%, respectively. Treatment with 5 mM dithioerythritol led to a heavy loss of activity and to dissociation into subunits (Mr 34000). Buthionine sulfoximine andL-methionine-sulfoximine, known as potent inhibitors of λ-glutamylcysteine synthetase from mammalian cells, were found to be effective inhibitors of the plant enzyme too. The apparent Km values forL-glutamate,L-cysteine, and α-aminobutyrate were, respectively, 10.4mM, 0.19 mM, and 6.36 mM. The enzyme was completely inhibited by glutathione (Ki=0.42 mM). The data indicate that the rate of glutathione synthesis in vivo may be influenced substantially by the concentration of cysteine and glutamate and may be further regulated by feedback inhibition of λ-glutamylcysteine synthetase by glutathione itself. λ-Glutamylcysteine synthetase is, like glutathione synthetase, localized in chloroplasts as well as in the cytoplasm. Chloroplasts fromPisum sativum L. isolated on a Percoll gradient contained about 72% of the λ-glutamylcysteine synthetase activity in leaf cells and 48% of the total glutathione synthetase activity. In chloroplasts ofSpinacia oleracea L. about 61% of the total λ-glutamylcysteine synthetase activity of the cells were found and 58% of the total glutathione synthetase activity. These results indicate that glutathione synthesis can take place in at least two compartments of the plant cell.

Glutathione in plants: biosynthesis and physiological role in environmental stress tolerance

“…The earliest step of GSH biosynthesis consists of a reaction between the c-carboxyl group of Glu and a-amino group of Cys to create an amide bond to yield c-ECS; and the c-ECS enzyme catalyzes this reaction. The next step is GSH formation; which occurs in the presence of the GSHS enzyme by amide bond formation between the a-carboxyl group of the cysteine moiety in cglutamylcysteine and the a-amino group of glycine, to form GSH (Hell and Bergmann 1990;Xiang and Oliver 1999). It is also possible to export the c-ECS from the plastids to the cytosol, where it serves as the precursor to GSH biosynthesis.…”

Oxidative Stimulation of Glutathione Synthesis in Arabidopsis thaliana Suspension Cultures

“…GSH synthesis is known to be regulated both by the supply of substrates, notably Cys, and also by feedback inhibition of 7-glutamylcysteine synthetase by reduced GSH (Rennenberg, 1982;Hell and Bergmann, 1990). Current models describing the stimulation of GSH levels in response to an oxidative stimulus propose that accumulation results from an overloading of the system with reactive oxygen species so that the capacity for GSH reductase to maintain GSH in the reduced form is exceeded and GSSG accumulates.…”

“…However, recent progress has established that in plants, as in a11 other organisms studied to date, GSH is synthesized by a two-step process. The first step, catalyzed by y-glutamylcysteine synthetase (Hell and Bergmann, 1990), results in the production of y-glutamylcysteine; the second, catalyzed by GSH synthetase, produces GSH through the addition of Gly (Rennenberg, 1982;Alscher, 1989).…”

Aγ-Glutamyl Transpeptidase-Independent Pathway of Glutathione Catabolism to Glutamate via 5-Oxoproline in Arabidopsis

“…First, g-glutamyl-Cys (g-EC) is synthesized by g-EC synthetase (Hell and Bergmann, 1990), and then Gly is incorporated by GSH synthetase (Wang and Oliver, 1996). The activity of g-EC synthetase is regulated by GSH levels (Hell and Bergmann, 1990;Jez et al, 2004).…”

“…First, g-glutamyl-Cys (g-EC) is synthesized by g-EC synthetase (Hell and Bergmann, 1990), and then Gly is incorporated by GSH synthetase (Wang and Oliver, 1996). The activity of g-EC synthetase is regulated by GSH levels (Hell and Bergmann, 1990;Jez et al, 2004). In Arabidopsis (Arabidopsis thaliana), g-EC synthetase and GSH synthetase are encoded by single genes, GSH1 (May and Leaver, 1994) and GSH2 (Wang and Oliver, 1996), respectively, and expression of both genes is regulated transcriptionally and translationally by heavy metals, jasmonic acid, and oxidative stress (Xiang and Oliver, 1998).…”