Tryptophanase mechanism

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. 

Since 1975 the structural genes of tryptophanase from E. coli K-12 and E. coli B/ It7-A have been isolated and sequenced, the primary structure of the enzyme determined, new catalytic intermediates detected, and single enzyme crystals suitable for X-ray analysis have recently been obtained. These and other studies on the structure and catalytic mechanism of tryptophanase are reviewed in this chapter. 

Fig. 9.5 Unfolding and refolding mechanism of E coli tryptophanase. See text for details.

The unfolding and refolding mechanism of E. coli tryptophanase is summarized in Fig. 9.5 (T. Mizobata and Y. Kawata, unpublished results), where N is the native state, I the intermediate having a tendency toward aggregation, X the irreversible aggregates, and U the unfolded state. At below 2 M Gdn-HCl, holotryptophanase unfolds via apoenzyme (Nhoio--- Napo-- I--> U (or X)), whereas at high concentrations of Gdn-HCl (>2 M),... 

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. 

Scheme XVII. Stereochemical mechanism of the tryptophanase reaction.

Gong F, Ito K, Nakamura Y, Yanofsky C. The mechanism of tryptophan induction of tryptophanase operon expression tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro). Proc. Natl. Acad. Sci. U.S.A. 2001 98 8997-9001. 

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. 

Protonation at the P-carbon to yield a protonated inline that is subsequently hydrolyzed to pyruvate and ammonia provides a reasonable mechanism for the final steps in the p-elimination reaction catalyzed by tryptophanase. BZ may bind preferentially to this gem-diamine species thereby stabilizing a labile reaction intermediate (structure 5B). 

As previously mentioned, tryptophanase is inhibited by a variety of different amino acids, which react with the PLP-cofactor to form covalent intermediates (102), but the structure of the substrate prevents completion of the reaction pathway. At equilibrium, these quasisubstrates generally form intense absorption bands in the 500-nm region of the spectrum, which result from the accumulation of a stable quinonoidal species. Phillips et al. (105) utilized RSSF in conjunction with SWSF studies to investigate the mechanisms of reaction for various aminoacid analogs of i-Trp in order to determine substrate structural elements important both for substrate binding and reactivity with the enzyme (structures 6-9). 

The mechanism of the p-elimination reaction catalyzed by tryptophanase is thought to require tautomerization of the indole ring to an activated indolenine intermediate with tetrahedral geometry at the C-3 carbon of the indole ring. Therefore, interactions between active-site residues and the ring nitrogen are likely to be very important for binding and catalysis. 

Since even a slightly elevated L-cysteine concentration is inhibitory or possibly toxic for the cells E. coli possesses another mechanism for detoxification of this compound in addition to excretion degradation of L-cysteine. Five enzymes with L-cysteine desulfhydrase activity have been identified so far in this organism L-tryptophanase (TnaA), L-cystathionine p-lyase (MetC),... 

Fig. 23. Mechanism of reaction of tryptophanase and tryptophan synthase. Reprinted from Reference 43 with permission of the American Chemical Society.

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... 

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