Evaluation of the Kinetic Mechanism of Escherichia coli Uridine Diphosphate-N-acetylmuramate:<scp>l</scp>-Alanine Ligase

Emanuele, John J.; Hu, Jin; Yanchunas, Joseph; Villafranca, Joseph J.

doi:10.1021/bi970266r

Cited by 58 publications

(58 citation statements)

References 17 publications

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“…ATP binding and active-site structural chemistry. Consistent with biochemical kinetic results that indicate that ATP is the first substrate to bind (12), ATP binding likely assembles the Mur ligases into an active conformation. The MurC ATPbinding site lies at the interface between the second and third domains with key interactions with the adenine ring and ␣-and ␤-phosphates provided by residues in the ATP-binding domain.…”

Section: Vol 185 2003supporting

confidence: 82%

Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:l-Alanine Ligase (MurC) fromHaemophilus influenzae

Mol¹,

Brooun²,

Dougan³

et al. 2003

J Bacteriol

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Section: Vol 185 2003supporting

confidence: 82%

Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:l-Alanine Ligase (MurC) fromHaemophilus influenzae

Mol¹,

Brooun²,

Dougan³

et al. 2003

J Bacteriol

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“…Assays containing ATP, ␥-glutamylcysteine, and either ␤-alanine, serine, or glutamate showed no enzymatic activity (10 and 50 mM substrate; 100 g AtGS). Initial Velocity Analysis of the Kinetic Mechanism-Initial velocity kinetic studies were performed by varying two of the three substrates while holding the third substrate constant under identical reaction conditions (24,25,28,29). Six families of data were generated such that for a saturating concentration of one substrate the reaction rates were measured over the entire range of the other two substrates.…”

Section: Resultsmentioning

confidence: 99%

“…These included six potential ordered mechanisms, nine possible partially ordered/random mechanisms, and a random kinetic mechanism. Computerized simultaneous fitting of the initial velocity data provides a robust analysis of the data compared with earlier reciprocal re-plotting approaches (24,29,30). Based on global fits to the initial velocity data, the best fit (r 2 ϭ 0.993) was obtained using the equation of a random Ter-reactant system (Equation 1).…”

Section: Resultsmentioning

confidence: 99%

Kinetic Mechanism of Glutathione Synthetase from Arabidopsis thaliana

Jez

Cahoon

2004

Journal of Biological Chemistry

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Glutathione synthetase (GS) catalyzes the ATP-dependent formation of the ubiquitous peptide glutathione from ␥-glutamylcysteine and glycine. The bacterial and eukaryotic GS form two distinct families lacking amino acid sequence homology. Moreover, the detailed kinetic mechanism of the bacterial and the eukaryotic GS remains unclear. Here we have overexpressed Arabidopsis thaliana GS (AtGS) in an Escherichia coli expression system and purified the recombinant enzyme for biochemical characterization. AtGS is functional as a homodimeric protein with steady-state kinetic properties similar to those of other eukaryotic GS. The kinetic mechanism of AtGS was investigated using initial velocity methods and product inhibition studies. The best fit of the observed data was to the equation for a random Ter-reactant mechanism in which dependencies between the binding of some substrate pairs were preferred. The binding of either ATP or ␥-glutamylcysteine increased the binding affinity of AtGS for the other substrate by 10-fold. Likewise, the binding of ATP or glycine increased binding affinity for the other ligand by 3.5-fold. In contrast, binding of either glycine or ␥-glutamylcysteine causes a 6.7-fold decrease in binding affinity for the second molecule. Product inhibition studies suggest that ADP is the last product released from the enzyme. Overall, these observations are consistent with a random Ter-reactant mechanism for the eukaryotic GS in which the binding order of certain substrates is kinetically preferred for catalysis.Glutathione is a key modulator of the intracellular reducing environment that provides protection against reactive oxygen species and maintains protein thiols in a reduced state (1). In plants, metabolic pathways for the detoxification of herbicides (2), air pollutants such as sulfur dioxide and ozone (3), and heavy metals (4) also rely on glutathione. For example, glutathione is the metabolic precursor for the synthesis of phytochelatin peptides that sequester cadmium, mercury, and arsenic (5).Glutathione biosynthesis occurs through a two-step pathway found in all organisms. In the first reaction, glutamate-cysteine ligase (EC 6.3.2.2) catalyzes the ATP-dependent formation of the dipeptide ␥-glutamylcysteine from cysteine and glutamate. Next, glutathione synthetase (GS, 1 EC 6.3.2.3) catalyzes the addition of glycine to the dipeptide (Scheme I).In this reaction, transfer of the ␥-phosphate group of ATP to the C-terminal carboxylate of ␥-glutamylcysteine yields an acylphosphate intermediate. Nucleophilic attack on the acylphosphate intermediate by glycine leads to formation of glutathione with release of ADP and inorganic phosphate (1, 6). GS from bacteria and eukaryotes form two distinct families that share no amino acid sequence homology (7). Detailed characterization of the Escherichia coli GS demonstrates that this enzyme is functional as a tetramer of ϳ300-residue subunits (8 -11), whereas the mammalian and yeast GS are active as dimers of ϳ470-residue subunits (12-15). Despite the lack of se...

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“…Analysis of the Kinetic Mechanism-Analysis of the kinetic mechanism of AtGCL⌬85 used global curve fitting (25,26). 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).…”

Section: Methodsmentioning

confidence: 99%

“…Kinetic Mechanism of AtGCL-To determine the kinetic mechanism of AtGCL, we obtained a complete matrix of kinetic data for the three substrates (25)(26)(27). Six families of data were generated in which one of the ligands is maintained at a saturating concentration whereas the other two substrates are varied.…”

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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Evaluation of the Kinetic Mechanism of Escherichia coli Uridine Diphosphate-N-acetylmuramate:l-Alanine Ligase

Cited by 58 publications

References 17 publications

Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:l-Alanine Ligase (MurC) fromHaemophilus influenzae

Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:l-Alanine Ligase (MurC) fromHaemophilus influenzae

Kinetic Mechanism of Glutathione Synthetase from Arabidopsis thaliana

Arabidopsis thaliana Glutamate-Cysteine Ligase

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