Escherichia coli gamma-glutamylcysteine synthetase. Two active site metal ions affect substrate and inhibitor binding.
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.
Kelly
Brenda S
BS
Department of Biochemistry and Biophysics Institute, Medical College of Wisconsin, Milwaukee, Wiscosin 53226, USA.
Antholine
William E
WE
Griffith
Owen W
OW
D001665:Binding Sites; D019328:Buthionine Sulfoximine / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*
D008345:Manganese; D019328:Buthionine Sulfoximine; D003300:Copper; D005721:Glutamate-Cysteine Ligase; D008274:Magnesium