Arabidopsis thaliana glutamate-cysteine ligase: functional properties, kinetic mechanism, and regulation of activity.
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.
Jez
Joseph M
JM
Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA. jjez@danforthcenter.org
Cahoon
Rebecca E
RE
Chen
Sixue
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D017360:Arabidopsis / Q000201:enzymology*; D001665:Binding Sites; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002850:Chromatography, Gel; D003538:Cystamine / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D025461:Feedback, Physiological; D015870:Gene Expression; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011487:Protein Conformation; D011994:Recombinant Proteins; D021241:Spectrometry, Mass, Electrospray Ionization
D004151:Dipeptides; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D018698:Glutamic Acid; D019328:Buthionine Sulfoximine; D000255:Adenosine Triphosphate; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003545:Cysteine; C017341:gamma-glutamylcysteine; D003538:Cystamine