Modeling the enzymatic transformation of 3,5-dimethoxy,4-hydroxy cinnamic acid by polyphenoloxidase from the white-rot fungus Trametes versicolor

A mechanism for transforming sinapic acid by a polyphenoloxidase from Trametes versicolor was investigated using changes in sinapic acid and oxygen concentrations during the reaction. The experiments were performed in a closed system without supplemental oxygen. The effects of temperature and initial oxygen concentration on the reaction rates were examined. To compare the obtained results with those from spectrophotometric studies, some runs were performed using an open system with supplemental oxygen. Sinapic acid transformation can be described by the Theorell-Chance Bi-Bi or Ordered Bi-Bi mechanisms. This reacting system consisted also of additional enzymatic reactions between the products of sinapic acid transformation and oxygen. A mathematical model was developed using four ordinary differential equations that represent the Theorell-Chance Bi-Bi mechanism with three alternate substrates. Model parameters (i.e., rate constants) were determined using the data collected at three different temperatures. On the basis of the transition state theory, relationships between these constants and temperature were established. It is shown that, in the open system, the observed change in the enzyme activity at higher temperatures was caused by two opposing phenomena: an Arrhenius effect which increased the rate, and a solubility effect which reduced the rate due to a lower oxygen concentration. This finding allows us to recommend better conditions for spectrophotometric methods, the assay most commonly used to evaluate this and similar enzymes. (c) 1996 John Wiley & Sons, Inc.