Protocatechuic acid oxidase

Protocatechuic acid is an intermediate in the metabolism of p-hydroxybenzoic acid by Vibrio species and by Pseudomonas fiuorescens (235,675,694,695). From the enzymically active dried cells (694) a protocatechuic acid oxidase has been extracted and partially purified... 

Protocatechuic acid oxidase prepared from Pseudomonas is highly specific. It does not attack catechol, hydroxyhydroquinone, 2,3-dihydroxybenzoio acid, 2,4-dihydroxybenzoic acid, the three mono-hydroxybenzoic acids, or various esters of protocatechuic acid, but some of these act as competitive inhibitors. Methylene blue cannot replace oxygen as an electron acceptor. 

Tracer studies of the fate of oxygen consumed during protocate-chuic acid cleavage have not been carried ocit, but because of oxygen stoichiometry, dependence of the enzyme upon ferrous ions, and resemblance of the over-all reaction to those catalyzed by pyrocate-chase, homogentisate oxidase, and 3-hydroxyanthranilate oxidase (compare 171), it is reasonable to classify protocatechuic acid oxidase as an oxygen transferase. 

The substrates of the polyphenol oxidase enzymes are phenolic compounds present in plant tissues, mainly flavonoids. These include catechins, anthocyanidins, leucoantho-cyanidins, flavonols, and cinnamic acid derivatives. Polyphenol oxidases from different sources show distinct differences in their activity for different substrates. Some specific examples of polyphenolase substrates are chlorogenic acid, caffeic acid, dicatechol, protocatechuic acid, tyrosine, catechol, di-hydroxyphenylalanine, pyrogallol, and catechins. 

From the cinnamic acids or phenyl propanoids described above, / -oxidation and truncation of side chains yields a variety of benzoic or simple phenolic acids [28], Rao et al., [22] identified gallic acid (18), gentisic acid (19), protocatechuic acid (20), />-hydroxybenzoic acid (21), oc-resorcyclic acid (22), vanillic acid (23) and salicylic acid (24) in C. arietinum and showed that overall, leaf content of all phenolic compounds was much greater than in roots and stem. They postulated that the production of these compounds may enhance the activity of indole acetic acid oxidase or may express antimicrobial properties when leached into the soil. However, Singh et al. [24] showed that the production of both 18 and 24 by C. arietinum was induced when treated by the culture filtrate of Sclerotium rolfsii along with the phenyl propanoids 14, 15 and 17 mentioned above. 

The phenol oxidase probably plays an important part in the sclerotization. We have purified the enzyme and have studied its action on various substrates. From these studies it appears that monophenols, including tyrosine, are only very slowly attacked by the enzyme. Dopa, dopamine and N-acetyldopamine are the best substrates of the diphenols tested. Other substances such as 3,4-dihydroxyphenylpyTuvicacid, 3,4-dihydroxyphenylacetic acid and protocatechuic acid are not attacked. 

Protocatechuic oxidase was found to be quite specific catechol, hydroxyhydroquinone, 2,3-dihydroxybenzoic acid, 2,4-dihydroxyben-zoic acid, o-, m-, and/ -hydroxybenzoic acids, and several esters of protocatechuic acid were not oxidized, although catechol and the dihydroxy-benzoic acids were found to be competitive inhibitors. Methylene blue failed to replace oxygen in this reaction. A similar enzyme induced in Neurospora by protocatechuic or vanillic acid also requires molecular oxygen and forms j8-carboxymuconic acid (Gross et al., 1956). 

Of a series of conventional inhibitors tested with the bacterial enzyme, only p-chloromercuribenzoic acid caused significant loss of activity, and this was prevented by glutathione. The Neurospora enzyme was not affected by iodoacetate, A -phenylmaleimide, semicarbazide, or cyanide (Ottey and Tatum, 1956). It is noteworthy that neither inhibition nor activation studies gave any evidence for a metal cofactor in the Pseudomonas preparation. An enzyme from Nocardia that is also inhibited by organic mercury also showed no evidence for a metal component (Cain and Cartwright, 1960). An iron requirement has been found for the Neurospora enzyme, however. When sufficiently pure preparations are available, it will be of interest to analyze the other protocatechuic oxidases for bound metal. 

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