The Effect of Oxidative Enzymes in Foods - Food Shelf Life Stability: Chemical, Biochemical, and Microbiological Changes - page 307

N

O N

O

Cu

Cu

(II)

(II)

N

R

N

FIGURE 9.3 Polyphenol oxidase peroxy complex. (From Zawistowski, J., Biladeris, C. G.,
and Eskin, N. A. M., Polyphenol oxidase, in Oxidative Enzymes in Foods, Robinson, D.S. and
Eskin, N. A. M., Eds., Elsevier Applied Science, London, 1991, Chap. 6. With permission.)

Through such a copper peroxide structure, Zawistowski et al. 125 have been able
to account for both the hydroxylation of monophenols and the dehydrogenation of
o-diphenols. It has been suggested that the hydroxylation or the dehydrogenation
reactions are determined by slightly different modes of binding of the monophenol
and the diphenol substrates to the peroxy form of the enzyme. Whether such geo-
metrical structures are universal for all polyphenol oxidases is unknown.

Assays and Inhibitors

For the hydroxylation reaction, tyrosine and p -coumaric acid are probably the natural
substrates, where tyrosine is converted to 3,4-dihydroxyphenylalanine (DOPA) and
p -coumaric acid is oxidized to caffeic acid. Chlorogenic acid (caffeoylquinic acid)
is formed by the hydroxylation of p -coumaroylquinic acid. A suitable test substrate
for the hydroxylation reaction is p -cresol. Oxygen uptake measured with an oxygen
electrode will measure both the hydroxylation and subsequently dehydrogenation
of the diphenol to form the quinone. Diphenolase activity can be assayed using
catechol or 4-methyl catechol as substrates. Initial rate measurements are best made
with the oxygen electrode, although other enzymes such as lipoxygenases, ascorbic
acid oxidase, and possibly peroxidases present in crude extracts may interfere. For
comparative measurements for different cultivars, the end product of the enzymic
reaction, the o -quinone, can be detected by its condensation with amino acids like
proline to form readily observed colored products. However, this end reaction is not
strictly quantitative, as the enzyme is irreversibly inhibited by its own generated
product, the o -quinone, naturally occurring reducing agents such as ascorbic acid
and thiols. Competitive inhibition by the substrate analogs cinnamic, p -coumaric,
and ferulic acids is possible. 127 There is a general trend towards avoiding the use of
chemical inhibitors, such as sulfite, and therefore an increasing effort is being made
to find further natural inhibitors 128 even from fungi and Lactobacillus species, some
of which have been reported to be low molecular weight peptides. 127 Effective
chemical inhibitors, especially for use in the laboratory for purification of PPOs are
cyanide, carbon monoxide, polyvinyl pyrrolidone (PVP), 4-hexyl-resorcinol, and
salicylhydroxamic acid (SHAM).
The closely related enzyme laccase 129 also contains Cu but may contain two or
four atoms per mole. Laccases are distinguished from polyphenol oxidases by their
ability to also catalyze the oxidation of p -diphenols as well as o -diphenols. The
enzyme is found in Basidiomycetes and fungi and was first discovered in the sap of