Redox‐Regulation of Glutathione Synthesis in Plants

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. Glutamate-cysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes; however, kinetic analysis shows that AtGCL is functionally similar to the enzyme from other organisms. Buthionine sulfoximine and cystamine inactivate AtGCL, but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. Global fitting analysis of initial velocity data indicates that a random ter-reactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consist of a catalytic subunit and a regulatory subunit, AtGCL functions as a monomeric protein. In response to changes in redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. Because reducing agents mediate this change, an intramolecular disulfide bond is likely involved. Thus, regulation of AtGCL does not require association with a regulatory subunit, as in the mammalian system, for activation. This model for controlling AtGCL activity explains observations that oxidative stimulus induces glutathione synthesis without increased gene expression in plants. Similar redox signal-induced conformational switching by disulfide formation occurs in other proteins, including Yap1, thioredoxins, and OxyR.