Role of aspartate 42 and histidine 79 in the aiPLA2 activity and oligomeric status of Prdx6 at low pH

Peroxiredoxin 6 (Prdx6), a unique non-seleno peroxidase, is a bifunctional protein with GSH peroxidase at pH 7.4 and calcium independent phospholipase A₂ (aiPLA₂) activities at pH 4.0. Changes in pH brings about alteration in the conformational and thermodynamic stability of Prdx6. For instance, under acidic condition (pH 4.0), Prdx6 forms higher oligomers with concommittant gain in aiPLA₂ activity that is resistant to thermal denaturation. However, there has been no molecular level understanding of how low pH induces formation of oligomers. In the present study, site directed mutagenesis of two conserved amino acid residues, Asp42 and His79, was used to study the molecular basis for the influence of pH on the oligomeric state of Prdx6. We observed that mutation at Asp42 and His79 residues by Ala did not result in a significant change in its peroxidase activity at neutral pH 7.4, but its aiPLA₂ activity at low pH 4.0 decreased significantly. At this pH condition, both mutants exhibit highly conserved alpha-helix content but fluctuating tryptophan micro-environment with partly exposed hydrophobic patches that render the formation of oligomers. DLS measurements and analytical SEC revealed that Wt Prdx6 forms oligomers at low pH but not the mutant proteins suggesting the importance of these residues in pH sensing and oligomerization. These results suggest that Asp42 and His79 interact each other to induce conformational change of Prdx6 that triggers the oligomerization of Prdx6 at low pH.