Assay, purification, properties and mechanism of action of γ-glutamylcysteine synthetase from the liver of the rat and <i>Xenopus laevis</i>

Davis, John S.; Balinsky, J.B.; Harington, J. S.; Shepherd, James

doi:10.1042/bj1330667

Cited by 36 publications

(14 citation statements)

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“…Several possible kinetic mechanisms have been proposed for mammalian ␥-GCS, including partially ordered ter-reactant (14 -16) and ping-pong mechanisms (17,18). Our data are not consistent with a ping-pong mechanism and supports the hypothesis that the substrates form a ternary complex on the enzyme prior to catalysis.…”

Section: Discussioncontrasting

confidence: 54%

“…This result is supported by the finding that buthionine sulfoximine is phosphorylated by ␥-GCS in the presence of ATP to form a tight binding transition state analog (12,13). Studies to delineate the kinetic mechanism of mammalian ␥-GCS have provided conflicting results; the reaction has been proposed to proceed through an ordered ter-ter mechanism (11), an ordered A (ATP) random BC mechanism (14,15), a random AB ordered C (L-Aba) mechanism (16), and various ping-pong mechanisms (17,18).…”

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confidence: 99%

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Trypanosoma brucei γ-Glutamylcysteine Synthetase

Brekken

Phillips

1998

Journal of Biological Chemistry

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The parasitic protozoan Trypanosoma brucei utilizes a conjugate of glutathione and spermidine, termed trypanothione, in place of glutathione to maintain cellular redox balance. The first committed step in the biosynthesis of glutathione and thereby trypanothione, is catalyzed by ␥-glutamylcysteine synthetase (␥-GCS). We have determined the kinetic mechanism for T. brucei ␥-GCS. The kinetics are best described by a rapid equilibrium random ter-reactant mechanism, in which the model derived K d values for the binding of L-Glu, L-␣-aminobutyrate, and ATP to free enzyme are 2.6, 5.1, and 1.4 mM, respectively. However, significant dependences exist between the binding of some of the substrate pairs. The binding of either ATP or L-Glu to the enzyme increases the binding affinity of the other by 18-fold, whereas the binding of L-Glu or L-␣-aminobutyrate decreases the binding affinity of the other by 6-fold. Similarly to the mammalian enzyme, cystamine is a time-dependent, irreversible inhibitor of T. brucei ␥-GCS. It has been suggested by several studies that cystamine labels an active site Cys residue essential for catalysis. Among the enzymes reported to be inactivated by cystamine, only one Cys residue is invariant (Cys-319 in T. brucei ␥-GCS). Mutation of Cys-319 to Ala in T. brucei ␥-GCS renders the enzyme insensitive to cystamine inactivation without significantly affecting the enzyme's catalytic efficiency, kinetic mechanism, or substrate affinities. These studies suggest that cystamine inactivates the enzyme by blocking substrate access to the active site and not by labeling an essential active site residue.

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Section: Discussioncontrasting

confidence: 54%

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confidence: 99%

Trypanosoma brucei γ-Glutamylcysteine Synthetase

Brekken

Phillips

1998

Journal of Biological Chemistry

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“…Kinetic Mechanism of AtGCL-The kinetic mechanism of GCL has been described as ping-pong (31,41), ordered A (ATP), random BC (42,43), or random ter-reactant (25) mechanisms. The kinetic data generated for AtGCL are not consistent with a ping-pong mechanism.…”

Section: Expression and Purification Of Atgcl-mentioning

confidence: 99%

Arabidopsis thaliana Glutamate-Cysteine Ligase

Jez

Cahoon

Chen

2004

Journal of Biological Chemistry

129

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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 ␥-glutamylcysteine from glutamate (K m ‫؍‬ 9.1 mM) and cysteine (K m ‫؍‬ 2.7 mM). Glutathione feedback inhibits AtGCL (K i ϳ1.0 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.Regulation of the intracellular redox environment is critical in cellular physiology for influencing signaling pathways and cell fate in response to stress (1). In plants, as in other organisms, glutathione plays multiple roles as protection against various environmental stresses (2). As an antioxidant, glutathione quenches reactive oxygen species and is involved in the ascorbate-glutathione cycle that eliminates peroxide (2). Plants use glutathione for the detoxification of xenobiotics (3), herbicides (4), air pollutants such as sulfur dioxide and ozone (5, 6), and heavy metals (7). Although glutathione accumulates in response to different stress stimuli in plants, the structural and kinetic properties of the plant enzymes responsible for its production remain biochemically uncharacterized.Glutathione synthesis occurs in two ATP-dependent steps. In the first reaction, glutamate-cysteine ligase (GCL) 1 (EC 6.3.2.2) catalyzes formation of the dipeptide ␥-glutamylcysteine from cysteine and glutamate (Scheme I). Addition of glycine to the dipeptide occurs in a second reaction, catalyzed by glutathione synthetase. Of the two enzymes, GCL appears to be rate-limiting (8). Exposure to heavy metals increases the levels of GCL mRNA in Brassica juncea and activates transcription of both GCL and glutathione synthetase in Arabid...

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“…y-Glutamylcysteine synthetase which catalyzes this reaction has been purified from rat liver [9], hog liver [lo], rat kidney [Ill, bovine and human erythrocytes [12,13] and bovine lens [14]. Lens y-glutaniylcysteine synthetase is quite specific for L-glutamate, but less specific for the L-cysteine moiety [14,1S].…”

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confidence: 99%

Bovine Lenticular γ‐Glutamylcysteine Synthetase: Reaction Sequence

Buskirk

Gander

Rathbun

1978

European Journal of Biochemistry

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The sequence of substrate addition and product release during the reaction catalyzed by y-glutamylcysteine synthetase was investigated with purified enzyme from bovine lens. Thermal inactivation and kinetic studies suggest that L-glutamate is the first substrate to bind to the enzyme. L-6-Chloroalanine was used as the L-cysteine analogue. Utilizing substrate activation and product inhibition studies, the following reaction sequence was determined : L-glutamate binding, ATP binding, ADP release, L-P-chloroalanine binding, followed by inorganic phosphate and then dipeptide release. The implications of this mechanism with regard to control of the enzyme in situ and its importance in glutathione synthesis are discussed.

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Assay, purification, properties and mechanism of action of γ-glutamylcysteine synthetase from the liver of the rat and Xenopus laevis

Cited by 36 publications

References 20 publications

Trypanosoma brucei γ-Glutamylcysteine Synthetase

Trypanosoma brucei γ-Glutamylcysteine Synthetase

Arabidopsis thaliana Glutamate-Cysteine Ligase

Bovine Lenticular γ‐Glutamylcysteine Synthetase: Reaction Sequence

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