Effect of protonation and aggregation state of (E)-resveratrol on its hydroperoxidation by lipoxygenase.
The protonation and aggregation states of (E)-resveratrol were used as tools to investigate the kinetic properties of lipoxygenase (LOX). It was found that the deprotonation of the 4'-hydroxyl group at pH values higher than the pK(a1) of (E)-resveratrol produced an increase in the LOX activity, with an optimum pH of 8.5. Moreover, the results show how LOX activity is strongly affected by the aggregation state of (E)-resveratrol. When the enzyme uses monomers of (E)-resveratrol as substrate, LOX shows a Michaelian behavior and the K(m) value can be determined (44.39 microM). However, when (E)-resveratrol concentration is increased to values higher than the critical concentration determined by fluorescence methods (35 microM at pH 8.5), LOX shows strong inhibition. These results can be interpreted as a previously unreported aggregate-induced enzyme inhibition, which can be modified by the use of different modulators of the aggregation state of (E)-resveratrol, such as cyclodextrins or ethanol. Finally, when the reaction was kinetically characterized in the optimum conditions of both aggregation and protonation state, a typical induction period was observed, along with a dependence of the hydroperoxidation rate with the hydrogen peroxide concentration.
Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Murcia, Spain. josemln@um.es
José Manuel
JM
López-Nicolás
Manuela
M
Pérez-Gilabert
Francisco
F
García-Carmona
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry*; D055601:Organic Chemistry Phenomena; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry*; D000077185:Resveratrol; D013025:Glycine max / Q000737:chemistry / Q000201:enzymology*; D013267:Stilbenes / Q000737:chemistry*
D010940:Plant Proteins; D013267:Stilbenes; D008084:Lipoxygenase; D000077185:Resveratrol