Identification of the Site for Phospholipid Binding to Peroxiredoxin 6: Coordination of the Phospholipase and Peroxidase Activities

Peroxiredoxin 6 (Prdx6) is the only member of the peroxiredoxin superfamily with both glutathione peroxidase (GPx) and phospholipase A2 (PLA2) activities. PLA2 activity of Prdx6 is abolished by “serine protease” inhibitors and inspection of its crystal structure suggests Ser32 – His 26 – Asp 140 as a catalytic triad. Site-specific mutation of S32 dramatically changed protein folding (by CD spectroscopy) making it difficult to evaluate the role of this amino acid residue on substrate binding and catalysis. H26A mutation had mimimal effect on the CD spectrum of Prdx6 indicating proper folding. This mutation abolished substrate (phosphatidylcholine) binding as determined by FRET analysis; PLA2 activity at pH 4 was 0.6 ± 0.2 for H26A vs. 102 nmol/min/mg prot for the wild type (WT). Peroxidase activity (reduction of H2O2) at pH 7 by the H26A mutant was 5600 ± 200 nmol/min/mg, similar to that of WT Prdx6. However, this mutation abolished catalytic reduction of peroxidized phospholipid as well as binding of the enzyme to peroxidized unilamellar liposomes. These results indicate that H26 in Prdx6 is part of the active site for binding of phospholipid substrate. The data also indicate that the PLA2 and phospholipid hydroperoxide peroxidase functions of Prdx6 share the site which controls enzyme – substrate binding as the initial step in catalysis, providing a mechanism for understanding linkage of these two activities. Research supported by HL19737.