Arabidopsis thaliana Glutamate-Cysteine Ligase: FUNCTIONAL PROPERTIES, KINETIC MECHANISM, AND REGULATION OF ACTIVITY

Joseph M Jez,Rebecca E Cahoon,Sixue Chen

doi:10.1074/jbc.m405127200

Joseph M Jez, Rebecca E Cahoon + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m405127200

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Abstract

In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamatecysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Because the putative Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes, the identity of this protein as a GCL has been debated. We have purified AtGCL from an Escherichia coli expression system and demonstrated that the recombinant enzyme catalyzes the ATP-dependent formation of gamma-glutamylcysteine from glutamate (Km = 9.1 mm) and cysteine (Km = 2.7 mm). Glutathione feedback inhibits AtGCL (Ki approximately 1.0 mm). As with other GCL, buthionine sulfoximine and cystamine inactivate the Arabidopsis enzyme but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. The slower inactivation rates observed with AtGCL suggest that the active site differs structurally from that of other GCL. Global fitting analysis of initial velocity data indicates that a random terreactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consists of a catalytic subunit and a regulatory subunit, AtGCL functions and is regulated as a monomeric protein. In response to redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. These results explain the reported posttranslational change in AtGCL activity in response to oxidative stress.

Highlights

In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione
Our results demonstrate that Arabidopsis thaliana GCL (AtGCL) shares functional properties with the Glutamatecysteine ligase (GCL) of other families but is regulated differently than the GCL of other non-plant eukaryotes or bacteria
Biochemical Evidence of GCL Function—Using purified recombinant protein, we have shown that AtGCL is a genuine GCL

Summary

EXPERIMENTAL PROCEDURES

Materials—Integrated DNA Technologies, Inc. synthesized all oligonucleotides used in this study. Enzyme activity was determined after addition of glutathione (0 –5 mM) to assay solutions containing either varied glutamate (1– 40 mM) or cysteine (0.5–20 mM). Inactivation data were plotted as log (% initial enzyme activity) versus time. Semilog plots were fitted to the equation ϪdE/dt ϭ k[I], where the disappearance of enzyme activity over time is related to the concentration of inactivator (I), multiplied by k, a rate constant. This allowed a determination of the half-life for inactivation (t1⁄2) at each [I]. The reaction rates were measured as described above using a matrix of substrate concentrations (glutamate, 2– 40 mM; ATP, 0.5–20 mM; cysteine, 1–20 mM). Synthesized ␥-glutamylcysteine was compared with an authentic standard (Sigma-Aldrich)

RESULTS

Substrate dependences

DISCUSSION