Tryptophanase Reaction

The presence of indole and skatole derivatives in urine and fecal contents has long been known. It was demonstrated by Hopkins and Cole that indole was formed from tryptophan by E. coli. 

The mechanism of the over-all reaction of indole formation was established by Woods, Gunsalus, and Umbreit, and was confirmed by Davis and Happold.  

Employing partially purified tryptophanase preparations from extracts of E. coli, it was demonstrated that the reaction sdelded indole, pyruvic acid, and NHj in approximately equimolar ratio, and that there was no uptake of oxygen. The enzyme preparation did not deaminate Stanier, R. Y., and Adelberg, E. A., Personal communications. 

208 Previous investigations of this subject led to the misleading results that oxygen was consumed and CO2 produced in the reaction also that riboflavin and DPN, as well as pyridoxal phosphate, were required for maximal activation. The confusion resulted from the fact that oxidation of pyruvate was being measured in the crude bacterial preparations as well as the decomposition of tryptophan to indole. For literature, see . 

The specificity requirements for this enzyme have been shown to be a free —COOH group, an unsubstituted —NHa group, a jS-carbon atom capable of oxidative attack, and an unsubstituted indole nitrogen atom. Beerstecher and Edmonds claimed that pyruvate and indole accelerated the reaction autocatalytically. The authors speculate that indole and pyruvate function by regenerating the coenzyme (pyridoxal phosphate) from its binding with tryptophan or tryptophan analogs. 

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Scheme XVII. Stereochemical mechanism of the tryptophanase reaction.

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). 

Although the tryptophan synthetase and tryptophanase reactions have been the best studied replacement and 0 eUmination-deamination reactions, others pf special interest are D-serine dehydratase [75-77] from E. coli, D-threonine dehydratase and L-threonine dehydratase from Serratia marcescens [78]. The only information available on the above enzymes is that in these cases also, the events at occur with retention of configuration. 

Fig. 26. Stereochemistry of the indolenine intermediates in the tryptophan synthase and tryptophanase reactions. Reprinted from Reference 44 with persmission of the American Society of Biological Chemists.

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). 

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. 

The Degradation of Tryptophan, Wood, Gunsalus, and Umbreit studied the tryptophanase reaction in extracts of E. coli. This enzyme was... 

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... 

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

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. 

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 ... 

A positive CD was found in the 500-nm quinonoid band which is formed on reaction of tryptophanase with L-alanine and oxindolyl-L-alanine (Fig. 9.10). The dissymmetry factor in this band is much smaller than in the absorption bands of the unliganded enzyme (Table 9.2). A negative 315-nm peak, which appears in the presence of L-alanine (Fig. 9.10), may be caused by interaction of an aromatic amino acid residue with the quinonoid coenzyme ring. 

It has been found68-70 that the reaction of tryptophanase with the inhibitory amino acids, /3-phenyl-DL-serine (threo form), L-threonine and D-alanine, is accompanied by a manifold increase in the reduced LD, i.e. in the ratio of LD to absorbance (AA / A) in the 420-425 nm band (Fig. 9.12 Table 9.2). This band belongs to the protonated internal PLP-lysine... 

General aspects of the stereochemistry and mechanism of PLP-catalyzed reactions have been reviewed by Floss and Vederas75 and Miles.77 In this section we briefly describe the catalytic cycle of tryptophanase. New transient intermediates have recently been detected in this cycle by Phillips et a/.,41-42-78 using rapid-scanning stopped-flow spectrophotometry, and they are included in the reaction mechanism depicted in Fig. 9.13. 

In the active site of the enzyme PLP forms an internal aldimine (Schiff base) with Lys-270 (Fig. 9.13,1). When the substrate is bound at the active site, its a-amino group attacks the C-4 atom of the coenzyme and replaces the -amino group of Lys-270 from its bond with PLP. This transaldimination reaction probably proceeds via a tetrahedral intermediate (a gem-diamine). Spectral evidence for formation of a gem-diamine in this step has recently been obtained in studies of the reaction of tryptophanase with L-homophenylalanine.41 The gem-diamine is subsequently converted to an external, PLP-substrate aldimine, and the -amino group of Lys-270 is released (Fig. 9.13, II). The equilibrium constant of this step with L-tryptophan is determined to be 11.6 mM.78 ... 

The closely related bacterial enzyme tyrosine phenol-lyase [137] has an even wider substrate and reaction specificity than tryptophanase, including the remarkable ability to cleave both D- and L-tyrosine and to interconvert D- and L-alanine. As already discussed and summarized in Tables 1 and 2, the stereochemistry at C-/J in all the a,/3-elimination and -replacement reactions of this enzyme studied so far is always retention [108,109,129]. This includes the a, -elimination of L- as well as of D-tyrosine. The fate of the a-hydrogen of L-tyrosine in this reaction has been probed in preliminary experiments (H. Kumagai, E. Schleicher and H.G. Floss, unpublished results), and the results tentatively suggest transfer of deuterium from the a-position to C-4 of the resulting phenol. Attempts to demonstrate intramolecularity of this transfer have so far been inconclusive. The base abstracting H-a in this enzyme may be histidine [138]. 

Krstulovic and co-workers (K30) reported on the development of assays for serum acid and alkaline phosphatase enzymes. These enzymes have been reported to be elevated in various disease states (A2, B14, F2, Kl). In addition, they developed a method for tryptophanase analysis using RPLC (K29). Employing fluorometric detection, the substrate, tryptophan, and reaction product, indole, can be monitored selectively with high sensitivity. They reported on several advantages of this method, including minimal sample preparation, rapid analysis time, and high specificity. 

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. 

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... 

This enzyme represents an interesting contrast to tryptophan synthase, which catalyzes the essentially irreversible formation of i-Trp. The spectrum of the native enzyme, which is highly pH dependent, is characterized by two absorbance bands centered at 420 nm and 337 nm. Early RSSF investigations utilizing rapid incremental jumps in pH showed that the two spectral bands arise from different protonation states of the covalently bound internal aldimine, E(Ain), form of the cofactor (101). Studies with a variety of amino acid inhibitors of tryptophanase (amino acids, which react reversibly with the enzyme to form covalent PLP-intermediates, but cannot complete the P-elimination reaction to form products), showed that the 420-nm species is the reactive form of the cofactor. The 337-nm species must be converted to the 420-nm species before reaction with the amino group of the substrate will occur. The 420-nm species represents aketoenamine form of the cofactor in which the iminium nitrogen of the Schiff s base is protonated (102). 

Fig. 16. Rapid-scanning data for the reactions of 5 mM indole (A) and 5 mM benzimidazole (B) with 17.2 iM tryptophan indole-lyase (tryptophanase) that has been preequilibrated with 0.25mM t-alanine. i-Alanine was premixed in both syringes to prevent unwanted concentration changes. In panel A, scans were collected at 15,92.5,170,247.5, 325,402.5,480,557.5, 635, 712.5, 790,867.5, and 945 ms after flow stopped. Spectrum 0 represents the spectrum of the enzyme-alanine complex before mixing with indole. In panel B, scans were collected at 15, 390, 765, 1140, 1515, 1890, 2265,2640, 3015,3390, 3765, and 4140 ms after mixing. Spectrum 0 represents the enzyme-alanine complex before mixing with benzimidazole. [Taken from Phillips (104) with permission.]...

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