title: Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7.
abstract: BACKGROUND
  Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.
  
  
  METHODS
  Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme-substrate and protein-protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.
  
  
  RESULTS
  Oxidation of the CP is fast (k+1>10(3)M(-1)s(-1)), however the rate of reduction by GSH is slow (k'+2=12.6M(-1)s(-1)) even though molecular docking indicates a strong GSH-GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+1>10(3)M(-1)s(-1)), but not by Trx. By surface plasmon resonance analysis, a KD=5.2μM was calculated for PDI-GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.
  
  
  CONCLUSIONS
  GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.
  
  
  CONCLUSIONS
  In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.
authors: Department of Molecular Medicine, University of Padova, Padua, Italy.
authors: Valentina
authors: V
authors: Bosello-Travain
authors: Marcus
authors: M
authors: Conrad
authors: Giorgio
authors: G
authors: Cozza
authors: Alessandro
authors: A
authors: Negro
authors: Silvia
authors: S
authors: Quartesan
authors: Monica
authors: M
authors: Rossetto
authors: Antonella
authors: A
authors: Roveri
authors: Stefano
authors: S
authors: Toppo
authors: Fulvio
authors: F
authors: Ursini
authors: Mattia
authors: M
authors: Zaccarin
authors: Matilde
authors: M
authors: Maiorino
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D005978:Glutathione / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005979:Glutathione Peroxidase; D006801:Humans; D051379:Mice; D062105:Molecular Docking Simulation; D009154:Mutation; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D002352:Carrier Proteins; C578349:NPGPx protein, mouse; D011993:Recombinant Fusion Proteins; D010544:Peroxidases; C558641:GPX7 protein, human; D005979:Glutathione Peroxidase; D019704:Protein Disulfide-Isomerases; D005978:Glutathione