Pyridoxal phosphate tryptophanase

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... 

Fig. 9.12 Effects of /3-phenyl-DL-scrine and L-threonine on absorption (A) and linear dichroism (B) spectra of gel-oriented tryptophanase in 100mM potassium phosphate buffer, pH 7.8. Curve I, unliganded enzyme. Curve 2, same as 1 + 165 mM L-threonine. Curve 3, same as 1 + 15 mM /3-phenyl-DL-serine threo form). (Reproduced with permission from Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors, p. 282, Pergamon, N. Y. (1991)).

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. 

Tyrosine is converted to phenol by an enzyme, /3-tyrosinase, studied especially by Japanese workers (456, 654, 658, 879). The enzyme, which has been partially purified, is inhibited by carbonyl reagents and is dependent on pyridoxal phosphate. The reaction is mechanistically probably (593) very similar to the tryptophanase reaction and is discussed when considering the function of pyridoxal phosphate (p. 91). 

Pyridoxal phosphate is the coenzyme in a large number of amino acid reactions. At this point it is convenient to consider together 1,he mechanism of those pyridoxal-dependent reactions concerned with aromatic amino acids. The reactions concerned are (1) keto acid formation (e.g., from kynurenine, above), 2) decarboxylation (e.g., of 5-hydroxytrypto-phan to 5-hydroxytryptamine, p. 106), (3) scission of the side claain (e.g., 3-tyrosinase, p. 78 tryptophanase, p. 110 and kynureninase, above), and 4) synthesis (e.g., of tryptophan from indole and serine, p. 40). Many workers have considered the mechanism of one or more of these reactions (e.g., 24, 216, 361, 595), but a unified theory is primarily due to Snell and his colleagues (summarized in 593). Snell s experiments have been carried out largely in vitro, and it should be emphasized that in vivo it is the enzyme protein which probably directs the electromeric changes. 

Scission of the side chain leaves an Ai ion which takes up a pioton to give indole from tryptophan (as with tryptophanase) or phenol from tyrosine (as with 3-tyrosinase). The side chain of the original molecule is left as the pyridoxal phosphate complex of aminoacrylic acid, and on hydrolysis the aminoacrylic acid tautomerizes to the imine of pyruvic acid which is hydrolyzed to pyruvic acid and ammonia ... 

Our laboratory has studied the stereochemistry of methyl group formation in a number of a, 0 elimination reactions of amino acids catalyzed by pyridoxal phosphate enzymes. The reactions include the conversions of L-serine to pyruvate with tryptophan synthase 02 protein (78) and tryptophanase (79), of L-serine and l-tyrosine with tyrosine phenol-lyase (80), and l-cystine with S-alkylcysteine lyase (81). In the latter study, the stereospecific isotopically labeled L-cystines were obtained enzymatically by incubation of L-serines appropriately labeled in the 3-position with the enzyme O-acetyl serine sulfhy-drase (82). The serines tritiated in the 3-position were prepared enzymatically starting from [l-3H]glucose and [l-3H]mannose by a sequence of reactions of known stereochemistry (81). The cysteines were then incubated with 5-alkyl-cysteine lyase in 2H20 as outlined in Scheme 19. The pyruvate was trapped as lactate, which was oxidized with K2Cr202 to acetate for analysis. Similarly, Cheung and Walsh (71) examined the conversion of D-serine to pyruvate with... 

Sodium borohydride reduction of the intermediates formed with other (nontransaminase) enzymes has given results very much in keeping with those found for aspartate aminotransferase. The tryptophan synthase (EC 4.2.1.20)/[4- H]pyridoxal-phosphate complex was reduced by NaBH4, both in the presence of the substrate serine and in its absence (43). Degradation of the products to pyridoxamine showed that, in the presence of substrate, reduction was from the 4 -Si face, whereas in its absence, reduction of the PLP-lysine imine was from the 4 -Re face (43). Tyrosine decarboxylase (EC 4.1.1.25) in the presence of tyrosine is reduced with NaB H from the 4 -Si face and in the absence of tyrosine from the 4 -Re face (44). Tryptophanase (EC 4.1.99.1) binds alanine as its Schiff base, which on reduction with NaB H and degradation was shown to be attacked at the 4 -Si face and at the a-carbon to yield (2S)-[2- H]alanine (45). 

Wood, W. A., I. C. Gunsalus and W. W. Umbreit Function of pyridoxal-phosphate Resolution and purification of the tryptophanase enzyme of Escherichia coli. J. biol. Chem. 170, 313 (1947). 

The tryptophanase reaction also requires pyridoxal phosphate, and differs from the synthetase reaction in that it forms pyruvate and ammonia as products, not serine (IV). 

The best method for the assay of pyridoxal phosphate is the use of tyrosine decarboxylase as described by Gunsalus, Bellamy, and Umbreit. The enzyme is prepared from a dried powder of cells of S. faecalis R. which has been grown deficient in vitamin Be by growth In a vitamin-Be-free alanine-rich medium. Thus, the decarboxylase is obtained almost completely resolved. This is a convenient preparation, since such a powder is stable for long periods and since the resolution of transaminases, decarboxylases, and tryptophanases isolated from tissues is a rather difficult task. The assay is performed manometrically by measuring the rate of CO2 liberation from tyrosine by the dried powder in the presence of pyridoxal phosphate. The rate of CO2 evolution is a function of the concentration of pyridoxal phosphate. 

Thus the mechanism of action of pyridoxal phosphate in transamination must be left open. There is as yet no clue to its mechanism of action in decarboxylation and in the tryptophanase reaction. 

Degradation of L-tryptophan takes place in some bacteria by the tryptophanase reaction in which the amino acid is converted to indole (35), pyruvic acid and ammonia. The reaction was first observed in 1903. Wood and his collaborators first prepared the enzyme tryptophanase which catalyses the change from Escherichia coli and showed that pyridoxal phosphate is the co-enzyme involved . Snell and his colleagues have proposed a mechanism for the reaction in which the required cleavage occurs via the intermediacy of a pyridoxal phosphate-tryptophan-metal complex... 

Various bacteria, E. coli in particular, cleave the side chain of tryptophan to yield indole S76). This putrefactive reaction is the source of indole and skatol in man. In earlier times these compounds were of great interest because they were believed to represent a serious health problem. Studies of this reaction with the enzyme tryptophanase has shown that indole, ammonia and pyruvic acid are the reaction products (377) and that the coenzyme for the reaction is pyridoxal phosphate... 

Other enzymes with pyridoxal phosphate moieties have been studied as well. Tryptophanase behaves similarly to cytoplasmic aspartate transaminase, whereas for serine hydroxymethyl transferase the P-NMR resonance is affected by pH but not by ligands (Schnackerz and Bartholmes, 1983 Quashnock et ai, 1983). 

Tryptophanase is an enzyme which catalyzes the stoichiometric interconversion of L-tryptophan and pyruvate, ammonia and indole it requires pyridoxal phosphate as a cofactor. The enzyme from Proteus rettgeri has been isolated in a crystalline state and its catalytic properties were investigated. The enzyme catalyzes a series of oc,p-elimination, P-replacement and the reversal of oc,P-elimination reactions. 

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

Tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) belongs to the family of the pyridoxal 5 -phosphate (PLP)-dependent enzymes. It serves in vivo to degrade L-tryptophan, is induced by L-tryptophan, and found in various bacteria, particularly in enteric species. Tryptophanase catalyzes a,/3-elimination1 and /3-replacement reactions on interaction with L-tryptophan and various other /3-substituted amino acids2 ... 

Pyridoxal 5 -phosphate (24), (25) Preparation, via affinity coupling, of active immobilized tryptophanase by treatment with apotryptophanase 30... 

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