Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background: Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C-P) it lacks the resolving Cys (C-R), 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 C-P is fast (k(+1) > 10(3) M-1 s(-1)), however the rate of reduction by GSH is slow (k'(+2) = 12.6 M-1 s(-1)) even though molecular docking indicates a strong GSH-GPx7 interaction. Instead, the oxidized C-P 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 K-D = 5.2 mu M was calculated for PDI-GPx7 complex. Participation of an alternative non-canonical C-R 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. General significance: In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH. (C) 2013 Elsevier B.V. All rights reserved.