Cooperative Binding of γ-Glutamyl Substrate to Human Glutathione Synthetase

Njålsson, Runa; Norgren, Svante; Larsson, Agne; Huang, Chin-Shiou; Anderson, Mary E.; Luo, Jiali

doi:10.1006/bbrc.2001.5961

Cited by 22 publications

(19 citation statements)

References 18 publications

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“…Unexpectedly, the purified enzyme was found to also catalyze the GS reaction. Since the GS specific activity is higher than reported for any previously purified GS (14,19,20), it is apparent that the 85-kDa protein rather than a trace impurity must account for the observed GS activity. Typical purifications using either auxiliary plasmid are summarized in Table I; the ␥-GCS/GS specific activity ratio was consistently 0.77 Ϯ 0.03 in several preparations.…”

Section: Identification and Cloning Of The S Agalactiae ␥-Gcs Gene Amentioning

confidence: 59%

“…c ND, not determined. d ␥-Glu-Cys and its analogs bind cooperatively to mammalian GS enzymes, and no specific K m value could be determined (14,20).…”

Section: Discussionmentioning

confidence: 99%

“…agalactiae ␥-GCS-GS exhibits a GS specific activity, determined under V max conditions, of 2383 Ϯ 66 units/mg (Table III), which is 2-fold higher than that of S. pombe GS (1206 Ϯ 18 units/mg) (23), about 4-fold higher than E. coli GS (650 units/ mg) (19) and about 7-fold higher than human GS (361 Ϯ 84 units/mg) (20). Table III also shows the K m values for the GS substrates and lists for comparison K m values for the E. coli, human, and rat GS enzymes.…”

Section: Studies Incubation Of Purified ␥-Gcs-gs With Atp L-glutamamentioning

confidence: 99%

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Glutathione Synthesis in Streptococcus agalactiae

Janowiak

Griffith

2005

Journal of Biological Chemistry

108

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␥-Glutamylcysteine synthetase (␥-GCS) and glutathione synthetase (GS), distinct enzymes that together account for glutathione (GSH) synthesis, have been isolated and characterized from several Gram-negative prokaryotes and from numerous eukaryotes including mammals, amphibians, plants, yeast, and protozoa. Glutathione synthesis is relatively uncommon among the Gram-positive bacteria, and, to date, neither the genes nor the proteins involved have been identified. In the present report, we show that crude extracts of Streptococcus agalactiae catalyze the ␥-GCS and GS reactions and can synthesize GSH from its constituent amino acids. The putative gene for S. agalactiae ␥-GCS was identified and cloned, and the corresponding protein was expressed and purified. Surprisingly, it was found that the isolated enzyme catalyzes both the ATP-dependent synthesis of L-␥-glutamyl-L-cysteine from L-glutamate and L-cysteine and the ATP-dependent synthesis of GSH from L-␥-glutamyl-L-cysteine and glycine. This novel bifunctional enzyme, referred to as ␥-GCS-GS, has been characterized in terms of catalytic activity, substrate specificity, and inhibition by GSH, cystamine, and transition state analog sulfoximines. The N-terminal 518 amino acids of ␥-GCS-GS (total M r 85,000) show 32% identity and 43% similarity with E. coli ␥-GCS (M r 58,000), but the C-terminal putative GS domain (remaining 202 amino acids) of ␥-GCS-GS shows no significant homology with known GS sequences. The C terminus (360 amino acids) is, however, homologous to D-Ala, D-Ala ligase (24% identity; 38% similarity), an enzyme having the same protein fold as known GS proteins. These results are discussed in terms of the evolution of GSH synthesis and the possible occurrence of a similar bifunctional GSH synthesis enzyme in other bacterial species.

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Section: Identification and Cloning Of The S Agalactiae ␥-Gcs Gene Amentioning

confidence: 59%

“…c ND, not determined. d ␥-Glu-Cys and its analogs bind cooperatively to mammalian GS enzymes, and no specific K m value could be determined (14,20).…”

Section: Discussionmentioning

confidence: 99%

Section: Studies Incubation Of Purified ␥-Gcs-gs With Atp L-glutamamentioning

confidence: 99%

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Glutathione Synthesis in Streptococcus agalactiae

Janowiak

Griffith

2005

Journal of Biological Chemistry

108

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“…Enzyme Assays and Kinetic Analysis-All kinetic analyses were done in duplicates using purified recombinant glutathione synthetase. The enzyme activity was measured at 37°C using a spectrophotometric assay, which couples ADP production to NADH oxidation and is monitored at 340 nm (38,39). In brief, the standard assay contained buffer (100 mM Tris-Cl, pH 8.2, 50 mM KCl, 20 mM MgCl 2 , 5 mM sodium phospho(enol) pyruvate, 0.2 mM NADH), 10 units of pyruvate kinase (Type III rabbit muscle), 10 units of lactic acid dehydrogenase (Type II rabbit muscle), and glutathione synthetase substrates (see below) (final volume of 0.2 ml) and was initiated by the addition of human glutathione synthetase.…”

Section: Methodsmentioning

confidence: 99%

“…Despite sharing the same function, the lack of sequence similarity between the two enzymes makes the previous studies of E. coli glutathione synthetase unsuitable for establishing the structure-function relationship of the human enzyme. Extensive kinetic studies of mammalian glutathione synthetase were carried out on the rat and recombinant human enzymes (38,39). The results suggested that there is a close catalytic dependence between the two substrates of the homodimer, generating a negative cooperativity for binding of ␥-glutamylcysteine substrate.…”

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confidence: 99%

Function of Conserved Residues of Human Glutathione Synthetase

Dinescu

Cundari

Bhansali

et al. 2004

Journal of Biological Chemistry

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Glutathione synthetase is an enzyme that belongs to the glutathione synthetase ATP-binding domain-like superfamily. It catalyzes the second step in the biosynthesis of glutathione from ␥-glutamylcysteine and glycine in an ATP-dependent manner. Glutathione synthetase has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. A variety of structural alignment methods were applied and four highly conserved residues of human glutathione synthetase (Glu-144, Asn-146, Lys-305, and Lys-364) were identified in the binding site. The function of these was studied by experimental and computational site-directed mutagenesis. The three-dimensional coordinates for several human glutathione synthetase mutant enzymes were obtained using molecular mechanics and molecular dynamics simulation techniques, starting from the reported crystal structure of human glutathione synthetase. Consistent with circular dichroism spectroscopy, our results showed no major changes to overall enzyme structure upon residue mutation. However, semiempirical calculations revealed that ligand binding is affected by these mutations. The key interactions between conserved residues and ligands were detected and found to be essential for enzymatic activity. Particularly, the negatively charged Glu-144 residue plays a major role in catalysis.

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Diagnostics in patients with glutathione synthetase deficiency but without mutations in the exons of the GSS gene

et al. 2003

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The synthesis of the ubiquitous tripeptide glutathione is impaired in patients with glutathione synthetase deficiency. The defect is inherited in an autosomal recessive manner, and the diagnosis is based on clinical, biochemical, and genetic criteria. In seven of our 30 index cases, however, no disease causing mutations could be identified in the coding exons or exon-intron boundaries of the glutathione synthetase gene GSS. These patients had severely decreased glutathione synthetase activities in lysates of cultured fibroblasts, and the levels of the enzyme were undetectable using a polyclonal antibody raised against human glutathione synthetase. RT-PCR mediated sequence analysis revealed previously not reported splice mutations in all patients. Thus, we conclude that in the investigation of patients with glutathione synthetase deficiency, and probably other genetic diseases as well, it might be time saving to initiate mutation analysis with sequencing of mRNA.

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Cooperative Binding of γ-Glutamyl Substrate to Human Glutathione Synthetase

Cited by 22 publications

References 18 publications

Glutathione Synthesis in Streptococcus agalactiae

Glutathione Synthesis in Streptococcus agalactiae

Function of Conserved Residues of Human Glutathione Synthetase

Diagnostics in patients with glutathione synthetase deficiency but without mutations in the exons of the GSS gene

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