The function of gamma-glutamylcysteine and bis-gamma-glutamylcystine reductase in Halobacterium halobium.
gamma-Glutamylcysteine and bis-gamma-glutamylcystine reductase appear to function in the halobacteria in a fashion analogous to GSH and glutathione reductase in other cells. Bis-gamma-glutamylcystine reductase (GCR), a NADPH-dependent dimer of Mr 122,000 recently purified to homogeneity from Halobacterium halobium (Sundquist, A.R., and Fahey, R.C. (1988) J. Bacteriol., 170, 3459-3467), was found to be highly specific for bis-gamma-glutamylcystine and to be present in cell extract at a level sufficient to maintain gamma-glutamylcysteine predominantly in its thiol form [( thiol]/[disulfide] approximately 50). Bis-gamma-glutamylcystine reductase is similar to glutathione reductase in many respects; GCR demonstrated a FAD:subunit stoichiometry of 1, inhibition by heavy metal ions, and a pH optimum near neutrality. However, GCR exhibited no activity with GSSG and was most active at salt levels exceeding 2 M. A turnover number of 1,700 mumol min-1 mumol-1 FAD and apparent Km values of 0.8 mM for bis-gamma-glutamylcystine and 0.29 mM for NADPH were determined for GCR. The effect of salt on the autoxidation rates of gamma-glutamylcysteine, GSH, and Cys was also studied. In the absence of added salt, Cys oxidized more rapidly than gamma-glutamylcysteine, which in turn oxidized more rapidly than GSH. The presence of 4.3 M chloride (K+ and Na+) significantly slowed the autoxidation of all three thiols. The rate of autoxidation of gamma-glutamylcysteine in 4.3 M chloride proved slower than that of GSH in the absence of added chloride. Thus, gamma-glutamylcysteine is at least as stable under halophilic conditions as GSH is under nonhalophilic conditions, explaining why halobacteria utilize gamma-glutamylcysteine rather than GSH.
Sundquist
A R
AR
Department of Chemistry, University of California-San Diego, La Jolla 92093-0506.
Fahey
R C
RC
D004151:Dipeptides / Q000378:metabolism*; D004220:Disulfides; D006217:Halobacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009994:Osmolar Concentration; D013379:Substrate Specificity
D004151:Dipeptides; D004220:Disulfides; D009247:NADH, NADPH Oxidoreductases; C056781:bis-gamma-glutamylcystine reductase; C017341:gamma-glutamylcysteine