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This paper reports a study on the hydroxylation of ferulic acid and tyrosine by field bean ( Dolichos
lablab) polyphenol oxidase, a reaction that does not take place without the addition of catechol. A
lag period similar to the characteristic lag of tyrosinase activity was observed, the length of which
decreased with increasing catechol concentration and increased with increasing ferulic acid
concentration. The activation constant K_a of catechol for ferulic acid hydroxylation reaction was 5
mM. The kinetic parameters of field bean polyphenol oxidase toward ferulic acid and tyrosine were
evaluated in the presence of catechol. 4-Methyl catechol, L-dihydroxyphenylalanine, pyrogallol, and
2,3,4-trihydroxybenzoic acid, substrates with high binding affinity to field bean polyphenol oxidase,
could stimulate this hydroxylation reaction. In contrast, diphenols such as protocatechuic acid, gallic
acid, chlorogenic acid, and caffeic acid, which were not substrates for the oxidation reaction, were
unable to bring about this activation. It is most likely that only o-diphenols that are substrates for the
diphenolase serve as cosubstrates by donating electrons at the active site for the monophenolase
activity. The reaction mechanism for this activation is consistent with that proposed for tyrosinase
(Sanchez-Ferrer, A.; Rodriguez-Lopez, J. N.; Garcia-Canovas, F.; Garcia-Carmona, F. Biochim.
Biophys. Acta 1995, 1247, 1-11). The presence of o-diphenols, viz. catechol, L-dihydroxyphenylalanine,
and 4-methyl catechol, is also necessary for the oxidation of the diphenols, caffeic acid, and
catechin to their quinones by the field bean polyphenol oxidase. This oxidation reaction occurs
immediately with no lag period and does not occur without the addition of diphenol. The kinetic
parameters for caffeic acid ( K_m ) 0.08 mM, V_max ) 32440 u/mg) in the presence of catechol and the activation constant Ka of catechol (4.6 mM) for this reaction were enumerated. The absence of a lag
period for this reaction indicates that the diphenol mechanism of diphenolase activation differs from
the way in which the same o-diphenols activate the monophenolase activity.