Tryptophan synthetase reactions

During my stay at Western Reserve University I also continued my studies of tryptophan synthetase—but now with the enzyme from E. coU as well as the one from Neurospora. With Jody Stadler and Martin Rachmeler initial observations were made that suggested that the tryptophan synthetase reaction was more complex than we had imagined. The enzyme converted indole-3-glycerol phosphate to indole and indole -3-glycerol phosphate to tryptophan in addition to catalyzing the previously characterized reaction, indole -F l-serine —> L-tryptophan< - - >. 

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... 

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. 

The conversion of indole to tryptophan has been much more extensively studied. This is brought about by direct reaction of indole and serine under the influence of the enzyme tryptophan desmolase (better named tryptophan synthetase) (302, 853, 854) which requires p5U-idoxal phosphate as a coenzyme (890). The enzyme has been obtained in the cell-free state (890) and partially purified (965) and its genetics in Neurospora studied in detail (966). Zinc appears to play some part in tryptophan desmolase formation or function (628). 

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 ... 

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. 

In the presence of both the substrates, indole-3-glycerolphosphate and L-serine, tryptophan synthetase, 02)82 catalyses two parallel reactions. The a subunits in the complex carry out the cleavage of indole-3-glycerolphosphate releasing glyceralde-hyde-3-phosphate in the medium but the indole remains bound to the complex. Concomitantly, the /Sj subunits promote the conversion of serine into the aminoacrylate-pyridoxal-P species (Fig. 27, 1) which acts as a Michael acceptor for the indole producing the L-tryptophan-pyridoxal-P complex from which the amino acid is released by hydrolysis (Fig. 27). That the aforementioned molecular events can be described in such vivid detail is due to an ingenious series of partial reactions... 

Fig. 27. A mechanism for the tryptophan synthetase, 2i82> catalysed reaction. The scheme shows that the indole formed in the reaction remains bound to the enzyme for condensation with the aminoacrylate intermediate.

Fig. 29. The retention of stereochemistry in the tryptophan synthetase catalysed reaction. The stereochemical assignment was made at the level of malate.

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. 

Studies with tryptophan synthetase [95] and tryptophanase [96] highlight the strength as well as the weakness of the spectroscopic approach as a tool for the identification and kinetic analysis of intermediary species produced in pyridoxal-P-dependent reactions. The involvement of the species of types 1, 2 and 4 (Fig. 31) in tryptophanase catalysed reactions and the kinetics of their formation have been extensively studied spectroscopically [96], but the quinonoid intermediate (2) and the acrylyl pyridinium species (4) could not be discerned from each other because the two chromophores absorbed in the same region at about 500 nm. 

Proteins associate with each other to form quaternary structures. Many proteins consist of more than one subunit. For example, hemoglobin has a molecular weight of 64,000 and is composed of four subunits, each of molecular weight 16,000. Two of the subunits are alike, and two are different. The enzyme tryptophan synthetase from Escherichia coli, which catalyzes the final two steps in the biosynthesis of that amino acid, consists of two nonidentical subunits, each of which catalyzes one reaction. Other enzymes contain regulatory and catalytic subunits. Still other enzymes consist of aggregates of two, three, or more identical subunits. The specific, noncovalent association of protein subunits is termed the quaternary stmcture of a protein. If the subunits are not identical, the association is called heterotypic. The association of identical subunits is termed homotypic. 

Describe the structure of tryptophan synthetase and the role of substrate channeling in its catalytic reaction. 

Reaction 3 is the cysteine desulfhydrase reaction which by -elimination produces HjS, pyruvate, and NH3. This reaction is catalyzed by cystathionine-y-lyase, the B protein of tryptophan synthetase (E.C. 4.2.1.20), or crystalline tryptophanase (E.C. 4.1.99.1) (Meister, 1965). According to Meister cysteine desulfhydrase reactions are probably catalyzed by other enzymes. Cystathionine-y-lyase has not been found in higher plants (Giovanelli et al., this volume. Chapter 12). 

Cystathionase also has a low level of direct cysteine desulphydrase activity. Tryptophanase and tryptophan synthetase are other enzymes capable of carrying out the cysteine desulphydrase reaction. Such considerations have cast doubt on this biological significance of this reaction, although strong arguments have been presented for a true cysteine desulphydrase in SalmonellcP. 

Fig. 3. Sephadex GlOO elution profiles showing the relative positions of yeast wild-type and trpS mutant enzyme activities. The first panel shows the relative positions at which the wild-type yeast tryptophan synthetase, assayed for reactions A and B, elutes from the column. The position at which E. coli A protein elutes is included for comparison. The nonsense mutants trS-3 and trS-9 gave the same elution profiles as tr5-35. The wild-type protein has a molecular weight of 160,000 and is excluded by the column. The fragment synthesized in nonsense mutants has a molecular weight of 35,000 and is retarded by the column.

The fifth enzyme activity, tryptophan synthetase [EC 4.2.1.20, L-serine hydro-lyase (adding indole)], catalyzes the final step in tryptophan synthesis. It consists of a complex of two nonidentical protein subunits, B (/ -chains) and A (a-chains) [7,14,38-41], which are coded for by the fourth and fifth tryptophan genes, respectively, in E. coli [14] and S. typhimurium [27]. The A subunit has been purified and shown to be a single polypeptide a-chain [42]. Wilson and Crawford [39] purified the B-protein subunit. Their conclusion that it was a dimer P2) which complexed with two a-chain subunits to form the tryptophan synthetase ( 2 Pi) is supported by other studies of the subunits and their association [40,41], The two proteins of tryptophan synthetase (TS) can catalyze the following reactions [38,43]. 

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