Monophenolase and diphenolase reaction mechanisms of apple and pear polyphenol oxidases

This paper reports a quantitative study of the effect of ring substituents on the rate of monophenol hydroxylation and o-diphenol oxidation catalyzed by apple and pear polyphenol oxidases. A good correlation between the electronic density at the carbon atom in the 4-position of the aromatic ring for each monophenol and the V-max values was found. However, this correlation was not so good in the 3- and 4-positions for the o-diphenols assayed. NMR studies on the monophenols demonstrated the higher reactivity of 4-hydroxyanisole compared with the other monophenols assayed. Catechol was the best o-diphenolic substrate assayed because of the absence of a ring substituent, All of these data confirmed the proposed enzyme's reaction mechanism and indicate that the rate-limiting step in the monophenolase reaction mechanism could be the nucleophilic attack of the oxygen atom belonging to the hydroxyl group at the carbon atom in the 4-position on the copper atoms of the enzyme's active site. However, in the diphenolase reaction mechanism, the rate-limiting step could be related to both the nucleophilic attack of the oxygen atom from the hydroxyl group at the carbon atom in the 3-position on the copper atoms of the enzyme's active site and the molecular size of the substituent side chain.