THEORETICAL SUPPORT FOR A CONFORMATIONAL CHANGE OF POLYPHENOL OXIDASE INDUCED BY METABISULFITE

摘要

Polyphenol oxidases (PPO) are responsible for the oxidation of phenols into quinones that give brown or black pigments. The aim of the work presented in this paper was to study the mechanism of inhibition of palmito (Acanthophoenix rubra) PPO by metabisulfite. When monitored spectrophotometrically and with an oxymeter, a decrease in enzymatic activity was observed. When measured with the oxymeter, there was a decrease in oxygen comsumption, indicating enzyme inhibition. When monitored with the spectrophotometer, the existence of a lag phase was noted, indicating that the quinones formed either are reduced into phenols or react with metabisulfite to give a colorless complex. The oxidation of phenol took place during the lag phase, and when metabisulfite was depleted, the amount of oxygen was not enough to allow the oxidation of the substrate in the optimal conditions. The plot of inhibition versus metabisulfite showed an allosteric behavior of PPO with positive cooperativity. The incubation of PPO with metabisulfite resulted in inhibition of enzymatic activity. This inhibition was as strong as the concentration of metabisulfite was high. The plot of log(residual activity) versus incubation time showed a diphasic behavior. All of these results indicate that PPO inhibition by metabisulfite does not obey a first-order reaction and that the action of metabisulfite is accompanied by a conformational change of the enzyme. Accordingly, bisulfite would react with the enzyme and form an intermediate complex that is more stable than the native form. The decomposition of this complex would give an inactive enzymatic form (Ei) through a slower second step. Fitting experimental values to the equation describing a two-step denaturation model confirmed the structural changes observed by electrophoresis by other authors.