3- Hydroxyanthranilic acid formation

Figure 9-53 Determination of lymphocyte kynureninase activity levels using HPLC. Enzyme activity is measured by quantification of formation of the product, 3-hydroxyanthranilic acid (3-HA A). (A) 3-HA A standard (12.0 nmol/L). (fl), Lymphocyte homogenate blank. (C) Lymphocyte 3-HAA production after 5 min of incubation in presence of 3-hydroxy-kynurenine. Peaks 1,3-HAA unmarked peaks are unidentified components. (From Ubbink et al., 1991.)...

The results with Neurospora led Bonner and Yanofsky (94) to suggest that the conversion of hydroxyanthranilic acid to nicotinic acid went by way of Intermediates A and B of diagram 21. Quinolinic acid formation was thought to be a shunt or side reaction of intermediate A, slow conversion to nicotinic acid possibly providing an alternative pathway. A similar conclusion was drawn from experiments in the rat (971), and it is now generally agreed that the conversion of quinolinic acid to nicotinic acid is at best of the order of a side reaction (e.g., 685, 754, and in man, 397, 696). 

An alternative route to nicotinic acid involves scission of hydroxyanthranilic acid between carbons 2 and 3, with intermediate formation of isocin-chomeronic acid (diagram 21). But the latter does not act as a nicotinic precursor in Neurospora (374), and this route can probably be excluded. 

Bokman AH, Schweigert BS (1951) 3-Hydroxyanthranilic acid metabolism. IV. Spectropho-tometric evi-dence for the formation of an intermediate. Arch Biochem Biophys 33 270-276... 

Purification has been reported of phenoxazinone synthase, the enzyme which catalyses phenoxazinone formation from 4-methyl-3-hydroxyanthranilic acid (123) in the biosynthesis of actinomycin (124). Two forms of the enzyme, one of high and one of low molecular weight, were isolated. The relative amount of the... 

Decarboxylation of 3-hydroxyanthranilic acid in the presence of picolinic carboxylase leads to the formation of picolinic acid. The steps involved in this transformation are not clear, nor are the enzymes involved known. 

Moline, S.W., Walker, H.C., Schweigert, B.S. 3-Hydroxyanthranilic acid metabolism. VII. Mechanism of formation of quinolinic acid. J. biol. Chem. 234, 880-883 (1959)... 

Henderson, L.M., and Ramasarma, G.B., 1949. Quinolinic acid metabolism. III. Formation from 3-hydroxyanthranilic acid by rat liver preparations. Journal of Biological Chemistry. 181 687-692. 

Fig. 34. Formation of oxidation product of 3-hydroxyanthranilic acid by indicated amounts of enzyme and subsequent nonenzymatic conversion to quinolinic acid. ...

Nicotinic Add Metabolism. The sequence of reactions leading to the formation of pyridine compounds is of particular interest as a source of nicotinic acid. Nutritional, isotopic, and genetic experiments have all shown that tryptophan and its metabolic derivatives including 3-hydroxy-anthranilic acid are precursors of nicotinic acid in animals and in Neuro-spora. The terminal steps in this sequence are not known. Under certain physiological conditions an increase in picolinic carboxylase appears to reduce nicotinic acid synthesis. This implies a common pathway as far as the oxidation of 3-hydroxyanthranilic acid. Whether quinolinic acid is a precursor of nicotinic acid is still uncertain. The enzyme that forms the amide of nicotinic acid also has not been isolated. Subsequent reactions of nicotinamide include the formation of the riboside with nucleoside phosphorylase and methylation by nicotinamide methyl-kinase. In animals W-methylnicotinamide is oxidized to the corresponding 6-pyridone by a liver flavoprotein. Nicotinic acid also forms glycine and ornithine conjugates. Both aerobic and anaerobic bacteria have been found to oxidize nicotinic acid in the 6-position. ... 

Pipkin, G. E., 1969, Inhibitory effect of L-ascorbate on tumor formation in urinary bladders implanted with 3-hydroxyanthranilic acid, Proc. Soc. Exp. Biol. Med. 131 522-524. 

Hydroxyanthranilic Acid. Since kynurenic and xanthurenic acids were eliminated as being precursors of nicotinic acid because tracer experiments showed that the alanyl side chain was lost prior to the formation of the vitamin, it appeared plausible to assume that 3-hydroxyanthranilic acid was an intermediate immediately following 3-hydroxykynurenine. In support of this, it has been found that it can replace niacin in the diet of the rat, but its activity is of the order of tryptophan rather than that of niacin. Furthermore, feeding 3-hydroxyanthranilic acid yields an increased urinary excretion of N -methyl nicotinamide. ... 

Quinolinic Acid. Assignment of a place to quinolinic acid as an intermediate in nicotinic acid formation is based on the evidence that its excretion into the urine is increased by injection of tryptophan or of 3-hydroxyanthranilic acid. Quinolinic acid isolated from the urine on treatment with acid gives rise to a substance having niacin activity for L. arabinosus. ... 

Other tracer experiments with N Mabeled 3-hydroxyanthranilic acid have shown that niacin and quinolinic acid are both derived quantitatively from this compound." The evidence which has been given above is in good agreement with the scheme for the formation of nicotinic acid given in Fig. 8. 

Y. Nishizuka, A. Ichiyama, R. K. Gholson, and O. Hayaishi, Studies on the metabolism of the benzene ring of tryptophan in mammalian tissues. I. Enzymic formation of glutaric acid from 3-hydroxyanthranilic acid, / Biol. Chem. 240, 733-739 (1965). 

Cleavage of the benzene ring of the hydroxyanthranilic acid moiety and conversion of the intermediate to nicotinic acid, picolinic acid, and quinolinic acid are very important steps in the metabolism of tryptophan. In addition to the formation of the above compounds, ring cleavage is part of the probable pathway for the complete oxidation of the hydroxyanthranilic acid to CO2 in vertebrates (323). 

In later work employing C -hydroxyanthranilic acid Mehler and May (334) demonstrated that the enzyme, picoUnic acid carboxylase, cleaves the carboxyl group of 3-hydroxyanthranilic acid in the process of picolinic acid formation. The enz3me is inhibited by CN and appears to require free —SH. The reactions leading to quinolinate and picolinate are shown in Fig. 20. 

In 1961, Priest el al. (6S) found that kidney and liver contiuned a soluble enzyme system which could promote the oxidation of 3-hydroxyanthranilic acid to quinolmic acid. Bokman and Schweigert (64) were able to demonstrate that an intermediate was involved in the oxidation. This intermediate can be characterized by an absorption maximum at 360 laix and has been referred to as compound 1, which spontaneously decomposes to quinolinic acid (66-67). Oxygen and ferrous ion are required for the formation of the intermediate (67), but not for its quantitative conversion to quinolinic acid. Compoimd I has been established by Wiss and Bettendorf (68) to be l-amino-4-formyl-l,3-butadiene-l,2-dicarboxylic acid. The enzyme 3-hydroxyanthranilic acid oxidase from beef liver has been purified slightly and evidence for the requirement of ferrous ion and sulfhydryl groups for activity has been obtained (68a). 

There is no evidence at present for the conversion of compound I to nicotinic acid. The formation of the vitamin from hydroxyanthranilic acid, however, has been demonstrated in rat liver slices and homogenates (71,72). The mechanism by which nicotinic acid is formed is not clear at present, although it is possible that the open chain saturated aldehyde shown in reactions (Ila) and (Ilia) in Fig. 2 may be an intermediate. The alpha decarboxylation and ring closure involved in the generation of nicotinic acid from compound I is not a spontaneous reaction and appears to be enzymic. Henderson (28) has pointed out that one of the reasons for the failure to observe nicotinic acid synthesis from compound I might be due to the competitive formation of quinolinic and picolinic acids. 

The primary oxidation product of 3-hydroxyanthranilic acid is the precursor of two pyridine compounds, quinolinic and picolinic acids (Mehler, 1956). The structure of the oxidation product is supported by this information, since a chemically plausible mechanism can be devised to acccount for these reactions. The formation of quinolinic acid is a nonenzymic, first-order reaction. The reaction rate is a function of temperature but not of the composition of the medium except for low pH. At low pH values a rapid, irreversible elimination of ammonia and carbon dioxide competes with quinolinic acid formation. At higher pH values the reaction may be considered as beginning with an isomerization of the double bond, leading to the formation of a compound with cu-carboxyl groups. This structure would ordinarily be the less favorable form. 

Cain RB (1968) Anthranilic acid metabolism by microorganisms. Formation of 5-hydroxyanthranilate as an intermediate in anthranilate metabolism by Nocardia opaca. Anthonie van Leewenhoek 34 417-432. 

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