Studies of peroxidase isolated from etiolated Alaska pea seedlings II. Effect of quercetin on the oxidation of indole-3-acetic acid

Abstract

1. 1. The effect of quercetin on the kinetic behaviour of Alaska pea peroxidase (H 2O 2 oxidoreductase, EC 1.11.1.7) at the time of oxidation of indole-3-acetic acid has been examined. 2. 2. Plots of reaction rates against indole-3-acetic acid concentration gave the usual hyperbolic and sigmoidal curves in the absence and presence of quercetin, respectively at pH 5.5. Apparent K m values and n values evaluated by Hill's plot were 4 · 10 −4 M and 1.9 in the presence of 1 · 10 −5 M quercetin, and 1.6 · 10 −2 M and 1.0 in its absence, respectively. 3. 3. Plots of reaction rates against quercetin concentration showed activation and inhibition patterns. The apparent Michaelis constant of activation (K a ) and n value were independent of indole-3-acetic acid concentration, giving values of 1 · 10 −4 M and 0.75, at pH 5.5, respectively. 4. 4. The effect of quercetin on the reaction rate was pH-dependent. Plots of the reaction rate against the indole-3-acetic acid concentration in the presence of 1 · 10 −5 M quercetin was hyperbolic at pH values lower than 4.5 with an n value of 1.0, whereas it was sigmoidal at a pH value higher than 4.5 with an n value greater than 1.0. 5. 5. Quercetin was degraded by peroxidase treatment with a maximal activity at pH 5.5, to a purple compound with an absorption maximum at 530 mμ. Indole-3-acetic acid inhibited the reaction non-competitively with an apparent inhibition constant (K i) of 1.5 · 10 −2 M at pH 5.5, which agreed well with the apparent K m for the indole-3-acetic acid oxidation in the absence of quercetin. 6. 6. KCN inhibited the reaction non-competitively with an apparent K i of 1 · 10 −3 M in the absence and presence of quercetin. 7. 7. The results obtained imply that: (a) each binding site of quercetin and indole-3-acetic acid is not that of heme; (b) the binding site of quercetin differs from that of indole-3-acetic acid. On the basis of kinetic analysis, two binding sites for indole-3-acetic acid were assumed, a catalytic site with a dissociation constant of 3 · 10 −4 M and an activating site with a dissociation constant of 2 · 10 − M in the presence of 1 · 10 −5 M quercetin at pH 5.5. This assumption can explain the experimental data well.