Kynureninase

Kynureninase is involved in the oxidative metabolism of tryptophan. It catalyzes the conversion of L-kynurenine to anthranilic acid. The enzyme also converts L-3-hydroxykyneurenine to 3-hydroxyanthranilic acid. The latter compound has a high fluorescence, which is the basis for detection in this assay. 

The reaction mixture was separated on a Whatman Partisphere C(8 column (4.6 mm x 150 mm, 5 /im). The mobile phase at a flow rate of 1 mL/min was 0.1 M potassium phosphate buffer (pH 5.8) containing 1% acetonitrile. The fluorescence detector was set for excitation and emission wavelengths of 322 and 414 nm, respectively. 

Homogenates of lymphocytes were preincubated at either 15 or 30°C for 10 minutes before the reaction was started, by addition of 3-hydroxykynure-nine to a final concentration of 1 mM. When the assay was performed at 30°C, the presence of 4 pM pyridoxal-5 -phosphate was needed to obtain a linear reaction. The reaction was terminated after 5 minutes by the addition of 150 piL of 10% trichloroacetic acid. HPLC analysis was carried out on 30 / L of the supernate obtained after centrifugation. 

Lymphocytes were isolated from human blood collected with EDTA as anticoagulant. Packed lymphocytes (10 pL) were resuspended in 225 pL of ice-cold sodium 5,5-diethylbarbiturate-HCL buffer (pH 8.4). After addition 

2 Kynureninase KynureninaseisapyridoxalphosphateivitaminBe)-dependent enzyme that catedyzes the hydrolysis of 3-hydroxykynurenine to 3-hydroxycmthranilic acid, releasing the side chain tis alanine. Impairment 

During the first half of the twentieth century, when 87,000 people died from pellagra in the United States, there was a two-fold excess of females over males. Reports of individual outbreaks of pellagra show a similar sex ratio. This may well be the result of inhibition of kynureninase, and impairment of the activity of kynurenine hydroxylase, by estrogen metabolites, and hence reduced synthesis of NAD from tryptophan (Bender and Totoe, 1984b). 

1 Kynurenine Hydroxylase Kynurenine hydroxylase is an FAD-dependent mixed-function oxidase of the outer mitochondrial membrane, which uses NADPH as the reductant. The activity of kynurenine hydroxylase in the liver of riboflavin-deficient rats is only 30% to 50% of that in control animals, and deficient rats excrete abnormally large amounts of kynurenic and anthranilic acids after the administration of a loading dose of tryptophan, and, correspondingly lower amounts of quinolinate and niacin metabolites. Riboflavin deficiency may thus be a contributory factor in the etiology of pellagra when intakes of tryptophan and niacin are marginal (Section 8.5.1). 

3-hydroxyanthranilic acid, releasing the side chain as tdcinine. Impairment 

This enzyme has been shown to occur in liver and kidney and also in certain microorganisms. It decomposes L-kynurenine to anthranilic acid and hydroxykynurenine to 3-hydroxyanthranilic acid. According to Wiss and Fuchs, the enzyme is able to split a variety of compounds containing the —COCH2CH(NH2)COOH group. Braunstein et observed that alanine was produced by the fission of the side chain and that a pyroxidine derivative was the coenzyme for the reaction. 

The formation of L-3-hydroxykynurenine as a tryptophan metabolite has been demonstrated in animals. This compound appears in the urine only in pyridoxine-deficient animals. 

Braunstein and co-workers demonstrated that pyridoxine deficiency did not affect the conversion of tryptophan to kynurenine. They showed that the kynureninase activity of the liver of pyridoxine-deficient animals was greatly reduced and could be restored in vitro by the addition of pyridoxal phosphate—a result that has been confirmed by Dalgliesh et According to Knox, removal of the alanyl side chain becomes the limiting step in the metabolism of tryptophan in pyridoxine deficiency and permits the accumulation of kynurenine, hydroxykynurenine, and their conversion products. Administration of extra tryptophan to the 

More recently Wiss found that kynureninase inactivated by dialysis could be reactivated by pyridoxal-5-phosphate, but not by pyridoxal-2-phosphate. As pyridoxine deficiency has no observable effect on the conversion of hydroxyanthranilic acid to nicotinic acid, the action of this vitamin appears to be connected solely with the removal of the alanyl side chain at the kynurenine level. 

---

Pyridoxamine phosphate serves as a coenzyme of transaminases, e.g., lysyl oxidase (collagen biosynthesis), serine hydroxymethyl transferase (Cl-metabolism), S-aminolevulinate synthase (porphyrin biosynthesis), glycogen phosphoiylase (mobilization of glycogen), aspartate aminotransferase (transamination), alanine aminotransferase (transamination), kynureninase (biosynthesis of niacin), glutamate decarboxylase (biosynthesis of GABA), tyrosine decarboxylase (biosynthesis of tyramine), serine dehydratase ((3-elimination), cystathionine 3-synthase (metabolism of methionine), and cystathionine y-lyase (y-elimination). 

Chlorodifluoromethylketones underwent aldol reactions (Eq. 124) via zinc enolates, to afford good yields of a,a-difluoro-/ -hydroxy ketones, in a study by the Kyoto group [327]. Copper(I) or silver salt catalysis was essential and boron-trifluoride additive appeared to exert a key role in the conversion to the enolate. Earlier [328], chlorodifluoromethyl ketones had been converted to the di-fluoroenoxy silanes by the action of zinc in the presence of chlorotrimethyl silane. A difluoroenoxy silane was used by McCarthy and co-workers [329] to synthesise a kynureninase inhibitor (Eq. 125) Lewis acid-mediated reaction with a chloroglycinate installed the key carbon-carbon bond. 

Returning to the major tryptophan catabolic pathway, marked by green arrows in Fig. 25-11, formate is removed hydrolytically (step c) from the product of tryptophan dioxygenase action to form kynurenine, a compound that is acted upon by a number of enzymes. Kynureninase (Eq. 14-35) cleaves the compound to anthranilate and alanine (step d), while transamination leads to the cyclic kynurenic acid (step e). Hie latter is dehydroxylated in an unusual reaction to quinaldic acid, a prominent urinary excretion product. 

Cleavage of 3-hydroxykynurenine by kynureninase (step g, Fig. 25-11) forms 3-hydroxyanthranilate, which is opened under the action of another dioxygenase (step h) with eventual degradation to acetyl-CoA, as indicated. In insects the reactive 3-hydroxyanthranilate is utilized in "tanning" reactions, e.g., coupling to tyrosine residues to toughen insect cuticles and walls of cocoons.214... 

Actinomycin.—Kynurenine and 3-hydroxykynurenine are actinomycin precursors in Streptomyces antibioticus (cf. Vol. 6, p. 42). Recently, kynureninase and hydroxykynureninase activity has been identified in S. parvulus cultures and the latter activity was found to show correlation with actinomycin formation.57... 

Although an earlier formulation had interpreted the kynureninase reaction in terms of an a,/5-elimination mechanism [142], the available evidence now points to a mechanism paralleling that of aspartate-/5-decarboxylase [141] as proposed by Braunstein [143]. No stereochemical studies have been reported on kynureninase, but some work has been done on aspartate-/5-decarboxylase. 

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.)...

Figure 8.4. Pathways of tryptophan metaholism. Tryptophan dioxygenase, EC 1.13.11.11 formylkynurenine formamidase, EC 3.5.1.9 kynurenine hydroxylase, EC 1.14.13.9 kynureninase, EC 3.7.1.3 3-hydroxyanthranilate oxidase, EC 1.10.3.5 picolinate carboxylase, EC 4.1.1.45 kynurenine oxoglutarate aminotransferase, EC 2.6.1.7 kynurenine glyoxylate aminotransferase, 2.6.1.63 tryptophan hydroxylase, EC 1.14.16.4 and 5-hydroxytryptophan decarboxylase, EC 4.1.1.26. Relative molecular masses (Mr) tryptophan, 204.2 serotonin, 176.2 kynurenine, 208.2 3-hydroxykynurenine, 223.2 kynurenic acid, 189.2 xanthurenic acid, 205.2 and quinolinic acid 167.1. CoA, coenzyme A.

Studies with [ C] tryptophan in animals and isolated hepatocytes show that leucine does inhibit the synthesis of NAD from tryptophan, inhibiting metabolism at the level of kynurenine hydroxylase and kynureninase, causing the accumulation of intermediates. In isolated hepatocytes, the more... 

A number of inborn errors of metabolism of the tryptophan oxidative pathway (see Figure 8.4) have been reported, aU of which result in the development of pellagra that responds to high doses of niacin. These conditions include vitamin Be-responsive xanthurenic aciduria, caused by a defect of kynureni-nase (Section 9.4.3) hydroxykynureninuria, apparentiy caused by a defect of kynureninase tryptophanuria, apparentiy caused by tryptophan dioxygenase deficiency a hereditary pellagra-like condition, apparentiy caused by an increase in activity of picoUnate carboxylase and Hartnup disease. 

Transamination Reactions of Other Pyridoxal Phosphate Enzymes Inaddition to theirmainreactions, anumberofpyridoxalphosphate-dependent enzymes also catalyze the half-reaction of transamination. Such enzymes include serine hydroxymethyltransferase (Section 10.3.1.1), several decarboxylases, and kynureninase (Section 8.3.3.2). 

Under normal conditions, the rate-limiting enzyme of the pathway is tryptophan dioxygenase (Section 8.3.2), and there is hide accumulation of intermediates. Kynurenine transaminase, the enzyme which catalyzes the transamination and ring closure of kynurenine to kynurenic acid, and of hydroxykynurenine to xanthurenic acid, has a high relative to the normal steady-state concentrations of its substrates in the liver. Kynureninase and kynurenine hydroxylase have lower values of K, so that there is normally litde accumuladon of kynurenine or hydroxykynurenine. 

As discussed in Section 8.3.3, estrogen metabolites inhibit kynureninase and reduce the activity of kynurenine hydroxylase to such an extent that, even without induction of tryptophan dioxygenase (Section 9.5.4.1), the activity of these enzymes is lower than is needed for the rate of flux through the pathway, thus leading to increased formation of xanthurenic and kynurenic acids. 

As discussed in Section 9.5.4.2, estrogen metabolites inhibit kynureninase, and they also lead to reduced activity of kynurenine hydroxylase. As a result, in pregnancy or in response to (high-dose) oral contraceptives, tissue concentrations of kynurenine, hydroxykynurenine, xanthurenic, and kynurenic acids are higher than normal. 

Kynureninase Kynureninase is a pyridoxal phosphate (vitamin Be) -dependent enzyme that catalyzes the hydrolysis of 3-hydroxykynurenine to... 

---