Tryptophan-2,3-dioxygenase

Tryptophan dioxygenase catalyzes the first step in the oxidative metabolism of L-tryptophan. L-Kynurenine is the product. 

L-Kynurenine was separated from 3-hydroxykynurenine (observed in some assays) by chromatography on a Beckman Ultrasphere ODS column (4.6 mm x 150 mm). The mobile phase (1.5 m/min) was a 5 235 mixture of acetonitrile and 0.1 M ammonium acetate buffer (pH 4.65). The column effluent was monitored at 365 nm. 

The reaction mixture in a 25 mL Erlenmeyer flask contained 1 mL of 0.2 M sodium phosphate (pH 7.0) and 0.075 M ascorbate, 100 / L of 42 ftM hematin HO (prepared in 0.01 M NaOH), 100 /xL of water, and 700 /xL of rat liver homogenate. After a 10-minute preincubation at 30°C, the reaction was started by adding 100 /xL of 40 mM L-tryptophan. At various times between 0 and 80 minutes, aliquots were removed and added to tubes containing 0.1 volume of 2.4 M perchloric acid. After chilling and centrifuging, then storage at room temperature for 2 hours to promote transformation of any formylkyneurine present to kyneurine, 25 /xL samples of each supemate were assayed. 

Liver tissue from rat was homogenized in 16 volumes of ice-cold normal saline by sonication. The whole homogenates were used in assay. 

The first enzyme of the pathway, tryptophan dioxygenase (also known as tryptophan oxygenase or tryptophanpyrrolase), is rate-limiting under normal conditions. In isolated hepatocytes, the control coefficient for flux through the pathway of tryptophan dioxygenase is 0.75 and that for tryptophan uptake into the cells is 0.25 (Salter et al., 1986). 

Tryptophan dioxygenase has a short half-life (of the order of 2 hours) and is subject to regulation by three mechanisms saturation with its heme cofactor, hormonal induction and feedback inhibition, and repression by NAD(P). 

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Tryptophan dioxygenase (indoleamine 2,3-dioxygenase)383 is a heme protein which catalyzes the reaction of Eq. 18-38. The oxygen atoms designated by... 

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. 

Figure 9.52 HPLC analysis of kynurenine in perchloric acid supernatants of tryptophan dioxygenase incubation mixtures. A sample of rat liver homogenate (containing 39 mg wet wt of tissue) was assayed for tryptophan dioxygenase. Aliquots of the assay mixture were removed at zero time and after 20,40, and 80 minutes of incubation at 30°C, and quenched by the addition of perchloric acid. Perchloric acid supernatants (25 /xL) were analyzed for kynurenine by HPLC. (From Holmes, 1988.)...

Livers from mice were manually sectioned into 1 to 1.5 mm cubes. In contrast to liver homogenates or purified enzyme, liver slices did not require exogenous methemoglobin or ascorbic acid for activation of tryptophan dioxygenase. 

Tryptophan dioxygenase (Section 8.3.2) is only found in the liver other tissues have an indoleamine dioxygenase, with lower specificity, that catalyzes the same reaction. However, the pathway for onward metabolism of kynure-nine is found only in liver and mononuclear phagocytes, and induction of indoleamine dioxygenase by cytokines, such as interferon-y, leads to increased circulating concentrations and urinary excretion of kynurenine, with litde or... 

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.

Saturation of Tryptophan Dioxygenase with Its Heme Cofactor... 

Induction of Tryptophan Dioxygenase by Glucocorticoid Hormones The de novo synthesis of tryptophan dioxygenase is induced by glucocorticoid hormones (cortisol in human beings and corticosterone in the rat). This is true induction of new mRNA and protein synthesis indeed. 

In isolated hepatocytes, after maximum induction of tryptophan dioxygenase by glucocorticoids, the uptake of tryptophan into the cells has a control coefficient of 0.75, whereas the control coefficient of tryptophan dioxygenase falls to 0.25. Therefore, the induction of tryptophan dioxygenase has only a limited effect on tryptophan catabolism and NAD synthesis (Salter and Pogson, 1985 Salter et al., 1986). In isolated perfused liver, although cortisol leads to a several-fold increase in tryptophan dioxygenase activity, there is only a relatively small increase in the rate of clearance of tryptophan from the perfusion medium (Kim and Miller, 1969). 

Even without induction of tryptophan dioxygenase, impairment of the activity of either enzyme may impair the onward metabolism of kynurenine and thus reduce the accumulation of aminocarboxymuconic semialdehyde and synthesis of NAD. 

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. 

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. 

Induction of extrahepatic mdoleamine dioxygenase (which catalyzes the same reaction as tryptophan dioxygenase, albeit by a different mechanism) by bacterial lipopolysaccharides and mterferon-y may result in the production of relatively large amounts of kynurenine and hydroxykynurenine in tissues that lack the enzymes for onward metabolism. Kidney has kynurenine transaminase activity, and therefore extrahepatic metabolism of tryptophan may result in significant excretion of kynurenic and xanthurenic acids, even when vitamin Bg nutrition is adequate. 

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. 

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