Tryptophan synthetase subunits

The two binding sites for indole on tryptophan synthetase subunits a and [3 are about 25 A apart. Explain how indole is transferred between these sites. 

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. 

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. 

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).

The above points show that a parallel can be drawn between the unfolding of globular proteins in solution and that at interfaces. A recent paper suggests that it can be carried on even on quantitative grounds. Comparison of the wild type and six mutant a-subunits of tryptophan synthetase substituted at the same position has evidenced correlations between their surface properties at air-water interface, linked to their interfacial unfolding, and their free energy of unfolding in solution [206]. 

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. 26. A stylised model of tryptophan synthetase, 2/ 2 the two components, a protein and P2 protein, derived therefrom. The model shows an intimate interaction between the a and fi subunits at the pyridoxal-P binding sites.

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. 

Tryptophan synthetase contains a 25-A-long channel between the active sites of adjacent a and (3 subunits. This channel allows the diffusion of the intermediate, indole, through the protein from one binding site to the other without diffusing away from the enzyme. This alleviates the potential problem of the hydrophobic indole molecule diffusing across the plasma membrane and out of the cell were it allowed to leave the enzyme. 

CREIGHTON, T.E., YANOFSKY, C., Association of the alpha and beta-2 subunits of the tryptophan synthetase of Escherichia coli, J. Biol. Chem., 1966,241, 980-990. 

Figure 5.41 Early steps of the proposed indole acetic acid biosynthesis pathways for Ara-bidopsis. CHO, chorismate ANA, anthranilate PANA, 5-phosphoribosylanthranilate CADP, l-(o-carboxyphenylamino)-l-deoxyribulose-5-phosphate IGP, indole-3-glycerol phosphate TRP, tryptophan. Enzymes ASA, anthranilate synthetase, suhunit a ASB, anthranilate synthetase, suhunit P PAT, phosphorihosylanthranUate transferase PAI, phosphoiibosylanthrani-late isomerase IGS, indole-3-glycerol-phosphate synthase TSA, tryptophan synthase, subunit a and TSB, tryptophan synthase, suhunit p.

Fig. 1. The tryptophan biosynthetic pathway. Abbreviations AS, anthranilate synthetase PRPP, 5-phosphoribosyI-l-pyrophosphate PRT, phosphoribosyi transferase PRAI, phosphoribosyi anthranilate isomerase InGPS, indoleglycerol phosphate synthetase TS, tryptophan synthetase TS-a, tryptophan synthetase a-chain subunit TS- z, tryptophan synthetase -chain dimer subunit.

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]. 

There is currently some uncertainty about the complexity of the tryptophan gene-enzyme relationships in B. subtilis. Whitt and Carlton [46,47] have noted pleiotropic effects. Most recently [46] they have found that the pleiotropy is limited to elimination of InGPS activity by mutations in either the trpD or trpF genes, which are primarily concerned with PRT and PRAI activity, respectively. The pleiotropic effects are shown in Fig. 3 by dotted lines and may indicate that the enzymes function as aggregates in vivo or may represent effects on translation similar to polarity effects. Hoch et al. [48], on the other hand, have not found these pleiotropic effects and report essentially one gene-one enzyme activity [when the individual activities of the tryptophan synthetase a and p2 subunits (Fig. 1) are included]. The nonconformity in the results of different investigators may be due to the use of different mutants and to different methods of preparation, affecting enzyme stabilities. 

Yanofsky, C., Drapeau, G. R., Guest, J. R. and Carlton, B. C. (1967) The complete amino acid sequence of the tryptophan synthetase A protein (a-subunit) and its colinear relationship with the genetic map of the A gene. Proc. Nat. Acad. Sci. 57, 296-298. 

Cistrons 1 and 2 correspond to A- and B-polypeptide subunits of tryptophan-synthetase, while cistrons 3A, 3B, and 4 determine the synthesis of enzymes converting anthranilic acid into indole-glycerophosphate. The order of these cistrons corresponds to the sequence of the synthetic reaction. 

Synthesis of tryptophan-synthetase is repressed by exogenous tryptophan, and derepression is observed if the tryptophan concentration in the medium is restricted. The A-protein subunit of tr5ptophan-synthetase may constitute, in the course of these variations, from 0.01 to l%of extractable cell protein (Yanofsky, 1960). 

A sequential unimolecular-bimolecular process was proposed to account for refolding and reactivation of tryptophan synthetase P2 subunit previously denatured in 4.5 M GuHCl at pH 2.3. The return of enzymatic activity can be described by first-order kinetics over a large concentration range (3-0.04 fiM) with a kinetic rate constant k = 6 1 x 10 " sec This was explained by a slow reshuffling process occurring after the first association... 

The structural and catalytic characteristics of tryptophan synthetase are under active investigation. The enzyme has a molecular weight of 135,000 and is composed of four subunits held together by noncovalent bonds. Floss and coworkers (361) determined the steric course of the tryptophan synthetase catalyzed reaction between serine and indole, using the enzyme isolated from Neurospora crassa. The reaction was... 

In oligomeric proteins composed of nonidentical subunits, it is possible to determine the functional role of each subunit, if dissociation and isolation of subunits are attained in their native states. Examples are tryptophan synthase, lactose synthetase, and aspartate transcarbamoylase.25 ... 

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