title: Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni.
abstract: Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.
authors.affiliation: Department of Microbiology and Immunology, Rush University Medical Center, Chicago, Illinois 60612, United States.
authors.forename: Hsin-Hung
authors.initials: HH
authors.lastname: Huang
authors.forename: Latasha
authors.initials: L
authors.lastname: Day
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Cass
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.forename: Charles H
authors.initials: CH
authors.lastname: Williams
authors.forename: David L
authors.initials: DL
authors.lastname: Williams
mesh_terms: D000818:Animals; D055162:Biocatalysis; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012550:Schistosoma mansoni / Q000201:enzymology*; D017279:Selenocysteine
chemical_list: D009097:Multienzyme Complexes; D017279:Selenocysteine; D009247:NADH, NADPH Oxidoreductases; C466433:thioredoxin glutathione reductase