title: Hysteresis in thioredoxin-glutathione reductase (TGR) from the adult stage of the liver fluke Fasciola hepatica.
abstract: Thioredoxin-glutathione reductase (TGR) was purified from the adult stage of the liver fluke Fasciola hepatica. At 38° C and pH 7.8, specific activity values were 10.2U mg(-1) and 64.5U mg(-1), with DTNB or GSSG as substrates, respectively. Under the same conditions, apparent Km values were 46±8 μM (DTNB) and 30 ± 5 μM (GSSG). The enzyme was also able to catalyze thiol/disulfide exchange reactions. A subunit Mr of 61,000 was obtained. Like the homologous enzyme from the tapeworms, a lag time was observed in the enzyme assays at moderate or high concentrations of the substrate GSSG. The hysteretic behavior was reverted in the presence of GSH and was notably dependent on pH, such that the magnitude of the lag time increased with the acidity of the medium. These results strongly suggest that a hysteretic kinetic is a common feature of TGR from any parasitic flatworm. A sequence comparison revealed the structural cysteine residues proposed to be in the origin of the peculiar kinetic behavior of TGR are absent from the F. hepatica enzyme. Based on these observations, the model proposed recently to explain the GSSG-dependent hysteretic kinetic of TGR, which assumes the covalent modification of specific cysteine residues through glutathionylation [Bonilla M. et al. (2008) J Biol Chem 283: 17898] needs to be reevaluated.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado Postal 70–159, 04510, DF México.
authors.forename: Alberto
authors.initials: A
authors.lastname: Guevara-Flores
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Pardo
authors.forename: Juan L
authors.initials: JL
authors.lastname: Rendón
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005210:Fasciola hepatica / Q000201:enzymology*; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016014:Linear Models; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D004220:Disulfides; D009097:Multienzyme Complexes; D013438:Sulfhydryl Compounds; D013879:Thioredoxins; D009247:NADH, NADPH Oxidoreductases; C466433:thioredoxin glutathione reductase; D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide