Abstract
We have shown previously and confirmed in this study that the phospholipase A(2) (PLA(2)) activity of peroxiredoxin 6 (Prdx6) is markedly increased by phosphorylation. This report evaluates the conformation and thermodynamic stability of Prdx6 protein after phosphorylation to understand the physical basis for increased activity. Phosphorylation resulted in decreased negative far-UV CD, strengthened ANS binding, and a lack of rigid tertiary structure, compatible with a change in conformation to that of a molten globule. The ΔG°(D) was 3.3 ± 0.3 kcal mol(-1) for Prdx6 and 1.7 ± 0.7 kcal mol(-1) for pPrdx6, suggesting that phosphorylation destabilizes the protein. Phosphorylation of Prdx6 changed the conformation of the N-terminal domain exposing Trp 33, as determined by tryptophan fluorescence and NaI fluorescence quenching. The kinetics of interaction of proteins with unilamellar liposomes (50:25:15:10 DPPC:egg PC:cholesterol:PG molar ratio) were evaluated with tryptophan fluorescence. pPrdx6 bound to liposomes with a higher affinity (K(d) = 5.6 ± 1.2 μM) than Prdx6 (K(d) = 24.9 ± 4.5 μM). By isothermal titration calorimetry, pPrdx6 bound to liposomes with a large exothermic heat loss (ΔH = -31.49 ± 0.22 kcal mol(-1)). Correlating our conformational studies with the published crystal structure of oxidized Prdx6 suggests that phosphorylation results in exposure of hydrophobic residues, thereby providing accessibility to the sites for liposome binding. Because binding of the enzyme to the phospholipid substrate interface is a requirement for PLA(2) activity, these results indicate that a change in the conformation of Prdx6 upon its phosphorylation is the basis for enhancement of PLA(2) enzymatic activity.