Escherichia coli gamma-glutamylcysteine synthetase. Two active site metal ions affect substrate and inhibitor binding

J Biol Chem. 2002 Jan 4;277(1):50-8. doi: 10.1074/jbc.M107961200. Epub 2001 Oct 23.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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*


  • Manganese
  • Buthionine Sulfoximine
  • Copper
  • Glutamate-Cysteine Ligase
  • Magnesium