Tryptophan synthetase

Tryptophan synthetase is the best known and most extensively studied example of the enzymes catalysing y8 replacement reactions. Our current understanding of the genetics and biochemistry of tryptophan synthetase is due to the painstaking and elegant studies of Yanofsky and coworkers [67] carried out during the last 2 decades at the University of Stanford. Tryptophan synthetase catalyses the last reaction in the biosynthesis of tryptophan  

Indole-3-glycerolphosphate + L-serine - L-tryptophan + D-glyceraldehyde-3-phosphate 

The enzyme is also capable of catalysing the following two half-reactions  

Indole-3-glycerolphosphate - indole -I- 3-glyceraldehyde-3-phosphate (5) Indole + L-serine - L-tryptophan (6) 

Tryptophan synthetase from E. coli contains two different types of polypeptides which are referred to as a and subunits. The physiologically functional tryptophan synthetase complex consists of two a and two y8 subunits and may be represented by the stylised illustration shown in Fig. 26. Upon dilution the complex dissociates to furnish the a subunits as monomers of mol. wt. 29 000 and a dimer consisting of two j8 subunits, mol. wt. 100000. The dimer contains one pyridoxal phosphate per polypeptide chain. These two components have been separated and used in the study of partial reactions. 

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Freedberg, W.B., and Hardman, J.K. (1971) Structural and functional roles of the cysteine residues in the a-subunit of the Escherichia coli tryptophan synthetase./. Biol. Chem. 246, 1439. 

Heilmann, H.D., and Holzner, M. (1981) The spatial organization of the active sites of the bifunctional oligomeric enzyme tryptophan synthetase Cross-linking by a novel method. Biochem. Biophys. Res. Comm. 99, 1146. 

Folding to native-like structure has been demonstrated with fragments of jS-galactosidase, lysozyme, serum albumin, penicillinase, and tryptophan synthetase. The capability of protein fragments for independent formation of structure therefore has substantial experimental basis. This generalization also makes plausible the idea that, in general, protein folding occurs by parts, that is, in a modular fashion. 

Most of the proteins on which fragment folding studies have been carried out are extracellular. Of the nine discussed in this article, only tryptophan synthetase and /3-galactosidase are not secreted. Many secreted proteins are synthesized with 20 or so additional amino acid residues at the N-terminus of the peptide chain (Blobel... 

Tryptophan synthetase from Escherichia coli is a simple example of a multienzyme complex It contains two types of subunits, and /3, that have molecular weights of 29,500 and 54,000, respectively.83-84 The fully associated enzyme has the composition a2/3285 and catalyzes the reaction... 

The coenzyme pyridoxal 5 -phosphate is required for the reactions in both (9) and (11). The sum of (10) and (11) gives (9), so that tryptophan synthetase is indeed a multienzyme complex catalyzing a sequence of reactions. The intermediate indole cannot be detected when the overall reaction is carried out, although the native enzyme will catalyze the partial reactions [(10) and (11)] 50 to 100 times more efficiently than the isolated subunits.86 88... 

Numerous equilibrium and kinetic studies have been made with tryptophan synthetase and its subunits, and considerable information has been obtained about the reaction pathway and reaction intermediates (cf. Refs. 89-92). For the purposes of this review, the principal conclusion reached is that the interaction of the a and j8 subunits appears to restrict the conformations of the a and /3 subunits to those that bind the substrates tightly and catalyze the reaction efficiently. The basic mechanism is not altered by the subunit interactions instead stabilization of particular conformations and binding sites is the important advantage gained in formation of the multienzyme complex. 

The pyruvate dehydrogenase complex from Escherichia coli is considerably more complex than tryptophan synthetase. It has a molecular weight of approximately 4.6 millon and contains three enzymes pyruvate dehydrogenase (Et), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3).82 The overall reaction catalyzed by the complex is... 

Schneider, W. P., Nichols, B. P., and Yanofsky, C. (1981). Procedure for production of hybrid genes and proteins and its use in assessing significance of amino acid differences in homologous tryptophan synthetase alpha polypeptides. Proc. Natl. Acad. Set. USA, 78, 2169-2174. 

Thus tryptophan synthetase, which catalyzes the addition of serine to indole via an QE.P-unsaturated imine derivative, is inactivated by QE-cyanoglycine (33). In this case, QE-cyanoglycine, an analogue of the substrate serine, undergoes Schiff base formation. Proton abstraction then occurs and the resultant QE-cyano carbanlon is apparently reprotonated to generate a reactive keteneimlne which can alkylate a nucleophilic active site residue. 

Fluorescence intensity of the pyridoxal phosphate group at the active site of tryptophan synthetase changes on addition of serine and indole, the substrates. 

Transamination is just one of a wide range of amino acid transformations that are catalyzed by PLP enzymes. The other reactions catalyzed by PLP enzymes at the a-carbon atom of amino acids are decarboxylations, deam-inations, racemizations, and aldol cleavages (Figure 23.12). In addition, PLP enzymes catalyze elimination and replacement reactions at the P-carbon atom (e.g., tryptophan synthetase Section 24.2.11) and the y-carbon atom (e.g., cytathionine P-synthase, Section 24.2.9) of amino acid substrates. Three common features of PLP catalysis underlie these diverse reactions. 

Many of the enzymes that catalyze these reactions, such as serine hy- droxymethyltransferase, which converts serine into glycine, have the same fold as that of aspartate aminotransferase and are clearly related by divergent evolution. Others, such as tryptophan synthetase, have quite different overall structures. Nonetheless, the active sites of these enzymes are remarkably similar to that of aspartate aminotransferase, revealing the effects of convergent evolution. 

Tryptophan Synthetase Illustrates Substrate Channeling in Enzymatie Catalysis... 

Tryptophan synthetase of E. coli, an a 2 P 2 tetramer, can be dissociated into two a subunits and a P 2 subunit (Figure... 

Figure 24.19. Structure of Tryptophan Synthetase. The structure of the complex formed by one a subunit and one P lb subunit. PLP is bound to the P subunit.

Figure 24.20. Substrate Channeling. A 25-A tunnel runs from the active site of the a subunit of tryptophan synthetase (yellow) to the PLP cofactor (red) in the active site of the P subunit (blue).

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