Phospholipid Hydroperoxides Are Substrates for Non-selenium Glutathione Peroxidase

Abstract

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl-or sn-2-arachidonoylphosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM ؊1 min ؊1 ) were k 1 ‫؍‬ 1-3 ؋ 10 5 and k 2 ‫؍‬ 4 -11 ؋ 10 4 . In the presence of 0.36 mM GSH, apparent K m was 120 -130 M and apparent V max was 1.5-1.6 mol/min/mg of protein. Assays with H 2 O 2 and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7-8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K i 4 M. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.

Peroxiredoxin, a Novel Family of Peroxidases

“…GSH has been suggested to be the physiological donor for 1-Cys Prx (10,11). However, several laboratories failed to detect GSH-supported peroxidase activity for 1-Cys-Prx (17,29).…”

Identification of a Novel Putative Non-selenocysteine Containing Phospholipid Hydroperoxide Glutathione Peroxidase (NPGPx) Essential for Alleviating Oxidative Stress Generated from Polyunsaturated Fatty Acids in Breast Cancer Cells

“…NPGPx, on the other hand, is unique because it incorporates regular cysteine at the catalytic center and shares structural similarity to the PHGPx from many different species (41,42). There is one other non-selenocysteine GPx (NSGPx) that has been described (12); however, in contrast to NPGPx, NSGPx (also known as 1-Cys peroxiredoxin) bears very little resemblance to the conserved motifs of GPx family members (53).…”

Adenovirus-mediated transfer of the 1-cys peroxiredoxin gene to mouse lung protects against hyperoxic injury

“…Of the six mammalian members of this family, five contain two reactive cysteines and utilize thioredoxin as the reductant (21). Peroxiredoxin VI or 1-cys peroxiredoxin (1-cysPrx) has a single redox-active cysteine and utilizes GSH to catalyze the reduction of H 2 O 2 and organic hydroperoxides including phospholipid hydroperoxides (8). 1-cysPrx is highly enriched in the lung compared with other organs and is especially expressed in Clara and alveolar epithelial type II cells (14).…”