PMID:11675389
| Citation |
Kelly, BS, Antholine, WE and Griffith, OW (2002) Escherichia coli gamma-glutamylcysteine synthetase. Two active site metal ions affect substrate and inhibitor binding. J. Biol. Chem. 277:50-8 |
|---|---|
| Abstract |
Gamma-glutamylcysteine synthetase (gamma-GCS, glutamate-cysteine ligase), which catalyzes the first and rate-limiting step in glutathione biosynthesis, is present in many prokaryotes and in virtually all eukaryotes. Although all eukaryotic gamma-GCS isoforms examined to date are rapidly inhibited by buthionine sulfoximine (BSO), most reports indicate that bacterial gamma-GCS is resistant to BSO. We have confirmed the latter finding with Escherichia coli gamma-GCS under standard assay conditions, showing both decreased initial binding affinity for BSO and a reduced rate of BSO-mediated inactivation compared with mammalian isoforms. We also find that substitution of Mn2+ for Mg2+ in assay mixtures increases both the initial binding affinity of BSO and the rate at which BSO causes mechanism-based inactivation. Similarly, the specificity of E. coli gamma-GCS for its amino acid substrates is broadened in the presence of Mn2+, and the rate of reaction for some very poor substrates is improved. These results suggest that divalent metal ions have a role in amino acid binding to E. coli gamma-GCS. Electron paramagnetic resonance (EPR) studies carried out with Mn2+ show that E. coli gamma-GCS binds two divalent metal ions; Kd values for Mn2+ are 1.1 microm and 82 microm, respectively. Binding of l-glutamate or l-BSO to the two Mn2+/gamma-GCS species produces additional upfield and downfield X-band EPR hyperfine lines at 45 G intervals, a result indicating that the two Mn2+ are spin-coupled and thus apparently separated by 5 A or less in the active site. Additional EPR studies in which Cu2+ replaced Mg2+ or Mn2+ suggest that Cu2+ is bound by one N and three O ligands in the gamma-GCS active site. The results are discussed in the context of the catalytic mechanism of gamma-GCS and its relationship to the more fully characterized glutamine synthetase reaction. |
| Links |
PubMed Online version:10.1074/jbc.M107961200 |
| Keywords |
Binding Sites; Buthionine Sulfoximine/metabolism; Copper/metabolism; Electron Spin Resonance Spectroscopy; Escherichia coli/enzymology; Glutamate-Cysteine Ligase/antagonists & inhibitors; Glutamate-Cysteine Ligase/chemistry; Glutamate-Cysteine Ligase/metabolism; Humans; Magnesium/metabolism; Manganese/metabolism |
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