Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A(2) activities.
Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A(2) (PLA(2)) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA(2) activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO2(3)) antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes.
Manevich
Yefim
Y
Institute for Environmental Medicine, University of Pennsylvania, 3620 Hamilton Walk, 1 John Morgan Building, Philadelphia, PA 19104-6068, USA.
Shuvaeva
Tea
T
Dodia
Chandra
C
Kazi
Altaf
A
Feinstein
Sheldon I
SI
Fisher
Aron B
AB
D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008081:Liposomes; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D054465:Peroxiredoxin VI / Q000378:metabolism*; D054467:Phospholipases A2 / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D008081:Liposomes; D010544:Peroxidases; D054465:Peroxiredoxin VI; D054467:Phospholipases A2