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Identification of the Site for Phospholipid Binding to Peroxiredoxin 6: Coordination of the Phospholipase and Peroxidase Activities
Author(s) -
Manevich Yefim,
Shuvaeva Tea,
Feinstein Sheldon I.,
Fisher Aron B.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a241-a
Subject(s) - chemistry , catalytic triad , phospholipase a2 , biochemistry , peroxiredoxin , active site , peroxidase , phospholipid , binding site , phospholipid hydroperoxide glutathione peroxidase , enzyme , serine protease , phosphatidylcholine , protease , glutathione peroxidase , glutathione , membrane
Peroxiredoxin 6 (Prdx6) is the only member of the peroxiredoxin superfamily with both glutathione peroxidase (GPx) and phospholipase A 2 (PLA 2 ) activities. PLA 2 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 S32A 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; PLA 2 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 H 2 O 2 ) 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 PLA 2 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.