Biomaterial Database

Escherichia coli gamma-glutamylcysteine synthetase. Two active site metal ions affect substrate and inhibitor binding. 2002

Brenda S Kelly, and William E Antholine, and Owen W Griffith

Department of Biochemistry and Biophysics Institute, Medical College of Wisconsin, Milwaukee, Wiscosin 53226, USA.

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.

UI	MeSH Term	Description	Entries
D008274	Magnesium	A metallic element that has the atomic symbol Mg, atomic number 12, and atomic weight 24.31. It is important for the activity of many enzymes, especially those involved in OXIDATIVE PHOSPHORYLATION.
D008345	Manganese	A trace element with atomic symbol Mn, atomic number 25, and atomic weight 54.94. It is concentrated in cell mitochondria, mostly in the pituitary gland, liver, pancreas, kidney, and bone, influences the synthesis of mucopolysaccharides, stimulates hepatic synthesis of cholesterol and fatty acids, and is a cofactor in many enzymes, including arginase and alkaline phosphatase in the liver. (From AMA Drug Evaluations Annual 1992, p2035)
D003300	Copper	A heavy metal trace element with the atomic symbol Cu, atomic number 29, and atomic weight 63.55.	Copper-63,Copper 63
D004578	Electron Spin Resonance Spectroscopy	A technique applicable to the wide variety of substances which exhibit paramagnetism because of the magnetic moments of unpaired electrons. The spectra are useful for detection and identification, for determination of electron structure, for study of interactions between molecules, and for measurement of nuclear spins and moments. (From McGraw-Hill Encyclopedia of Science and Technology, 7th edition) Electron nuclear double resonance (ENDOR) spectroscopy is a variant of the technique which can give enhanced resolution. Electron spin resonance analysis can now be used in vivo, including imaging applications such as MAGNETIC RESONANCE IMAGING.	ENDOR,Electron Nuclear Double Resonance,Electron Paramagnetic Resonance,Paramagnetic Resonance,Electron Spin Resonance,Paramagnetic Resonance, Electron,Resonance, Electron Paramagnetic,Resonance, Electron Spin,Resonance, Paramagnetic
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005721	Glutamate-Cysteine Ligase	One of the enzymes active in the gamma-glutamyl cycle. It catalyzes the synthesis of gamma-glutamylcysteine from glutamate and cysteine in the presence of ATP with the formation of ADP and orthophosphate. EC 6.3.2.2.	gamma-Glutamyl-Cysteine Synthetase,Glutamylcysteine Synthetase,Glutamate Cysteine Ligase,Ligase, Glutamate-Cysteine,Synthetase, Glutamylcysteine,Synthetase, gamma-Glutamyl-Cysteine,gamma Glutamyl Cysteine Synthetase
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D019328	Buthionine Sulfoximine	A synthetic amino acid that depletes glutathione by irreversibly inhibiting gamma-glutamylcysteine synthetase. Inhibition of this enzyme is a critical step in glutathione biosynthesis. It has been shown to inhibit the proliferative response in human T-lymphocytes and inhibit macrophage activation. (J Biol Chem 1995;270(33):1945-7)	Sulfoximine, Buthionine