Tryptophanase

Tryptophanase catalyzes the conversion of tryptophan to indole and acetic acid. Pyridoxal phosphate is a required cofactor. The HPLC method developed to assay this activity involves the separation of the tryptophan from the indole. 

The separation was carried out by reversed-phase HPLC (Ci8 /uBondapak) using a mobile phase of anhydrous methanol-water (1 1, v/v). The column was 

The reaction mixture contained potassium dihydrogen phosphate buffered to pH 7.0 and bacterial cells. The cells were sonicated and preincubated with pyridoxal phosphate, and the reaction was started by the addition of 

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L-tryptophan indole + pymvic acid +NH tryptophanase Proteus rettgerii ... 

FIGURE 10.3 Pathways for degradation of L-tryptophan by (a) tryptophanase, (b) deamination and oxidation, and (c) side-chain oxidation and decarboxylation to indole. 

Hoch SO N, RD DeMoss (1972) Tryptophanase from Bacillus alvei. 1. Subunit structure. J Biol Chem 7A1 1750-1756. 

Kamath AV, C Yanofsky (1992) Characterization of the tryptophanase operon in Proteus vulgaris. J Biol Chem 267 19978-19985. 

Morino Y, EE Snell (1967) A kinetic study of the reaction mechanism of tryptophanase-catalyzed reactions. J Biol Chem 242 2793-2799. 

Newton WA, Y Morino, EE Snell (1965) Properties of crystalline tryptophanase. J Biol Chem 240 1211-1218. 

Vederas JC, E Schleicher, M-D Tsai, HG Floss (1978) Stereochemistry and mechanism of reactions catalyzed by tryptophanase from Escherichia coli. J Biol Chem 253 5350-5354. 

Woolridge, E. M. Rokita, S. E. 6-(Difluoromethyl)tryptophan as a probe for substrate activation during the catalysis of tryptophanase. Biochemistry 1991, 30, 1852-1857. 

Pyridoxal Phosphate.—Analogues of pyridoxal and pyridoxamine 5 -phosphates have frequently been used to probe the size and shape of the active sites of a number of enzymes. For example, the apoenzyme of a tryptophanase from Bacillus alvei will bind pyridoxal 5 -phosphate as well as the 2-nor, 2 -methyl, 2 -hydroxy, 6-methyl, and A-oxide analogues.27 No analogue that has been modified at C-4 binds to the enzyme, confirming the absolute requirement for Schiff-base formation between the... 

THREONINE DEHYDRATASE TRYPTOPHANASE TYROSINE AMINOTRANSFERASE Pyridoxal S -phosphate, synthesis of, PYRIDOXAL KINASE... 

ACYL-SERINE INTERMEDIATE CHYMOTRYPSIN CATALYTIC TRIAD TRYPTOPHANASE TRYPTOPHAN SYNTHASE T state,... 

Tryptophanase-indu- zierte immobilisierte E.-CoLi Zcllen auf Polyacry lam i d-G el PLPb OcJ /nhj 40 4... 

Test for indole Indole is a component of the amino acid tryptophan, which can he broken down by the bacterial enzyme tryptophanase. When tryptophan is broken down, the presence of indole can be detected through the use of Kovacs reagent. Kovacs reagent, which is yellow, reacts with indole and produces a red colour on the surface of the test tube. Kovacs reagent is prepared by dissolving 10 g of p-aminobenzaldehyde in 150 mL of isoamylalcohol and then slowly adding 50 mL of concentrated HCl. 

Hall, A.N. et al., The degradation of some benzene substituted tryptophans by Escherichia coli tryptophanase, Biochem.., 74, 209, 1960. 

J. London, C. Skrzynia, and M. E. Goldberg, Renaturation of Escherichia cdi tryptophanase after exposure to 8 M urea. Evidence for the existence of nucleation centers, Eur.J. Biochem. 1974, 47, 409-415. 

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