Allosteric interactions coordinate catalytic activity between successive metabolic enzymes in the tryptophan synthase bienzyme complex.

Tryptophan synthase from enteric bacteria is an alpha 2 beta 2 bienzyme complex that catalyzes the final two reactions in the biosynthesis of L-tryptophan (L-Trp) from 3-indole-D-glycerol 3'-phosphate (IGP) and L-serine (L-Ser). The bienzyme complex exhibits reciprocal ligand-mediated allosteric interactions between the heterologous subunits [Houben, K., & Dunn, M. F. (1990) Biochemistry 29, 2421-2429], but the relationship between allostery and catalysis had not been completely defined. We have utilized rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy to study the relationship between allostery and catalysis in the alpha beta-reaction catalyzed by the bienzyme complex from Salmonella typhimurium. The pre-steady-state spectral changes that occur when L-Ser and IGP are mixed simultaneously with the alpha 2 beta 2 complex show that IGP binding to the alpha-site accelerates the formation of alpha-aminoacrylate [E(A-A)] from L-Ser at the beta-site. Through the use of L-Ser analogues, we show herein that the formation of the E(A-A) intermediate is the chemical signal which triggers the conformational transition that activates the alpha-subunit. beta-subunit ligands, such as L-Trp, that react to form covalent intermediates at the beta-site, but are incapable of E(A-A) formation, do not stimulate the activity of the alpha-subunit. Titration experiments show that the affinity of G3P and GP at the alpha-site is dependent upon the nature of the chemical intermediate present at the beta-active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Allosteric regulation of substrate channeling in tryptophan synthase: modulation of the L-serine reaction in stage I of the beta-reaction by alpha-site ligands

In the tryptophan synthase bienzyme complex, indole produced by substrate cleavage at the alpha-site is channeled to the beta-site via a 25 A long tunnel. Within the beta-site, indole and l-Ser react with pyridoxal 5'-phosphate in a two-stage reaction to give l-Trp. In stage I, l-Ser forms an external aldimine, E(Aex1), which converts to the alpha-aminoacrylate aldimine, E(A-A). Formation of E(A-A) at the beta-site activates the alpha-site >30-fold. In stage II, indole reacts with E(A-A) to give l-Trp. The binding of alpha-site ligands (ASLs) exerts strong allosteric effects on the reaction of substrates at the beta-site: the distribution of intermediates formed in stage I is shifted in favor of E(A-A), and the binding of ASLs triggers a conformational change in the beta-site to a state with an increased affinity for l-Ser. Here, we compare the behavior of new ASLs as allosteric effectors of stage I with the behavior of the natural product, d-glyceraldehyde 3-phosphate. Rapid kinetics and kinetic isotope effects show these ASLs bind with affinities ranging from micro- to millimolar, and the rate-determining step for conversion of E(Aex1) to E(A-A) is increased by 8-10-fold. To derive a structure-based mechanism for stage I, X-ray structures of both the E(Aex1) and E(A-A) states complexed with the different ASLs were determined and compared with structures of the ASL complexes with the internal aldimine [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Biochemistry 46, 7713-7727].     

Synergistic effects on escape of a ligand from the closed tryptophan synthase bienzyme complex

Substrate channeling in the tryptophan synthase bienzyme complex is regulated by allosteric signals between the alpha- and beta-active sites acting over a distance of 25 A. At the alpha-site, indole is cleaved from 3-indole-D-glycerol 3'-phosphate (IGP) and is channeled to the beta-site via a tunnel. Harris and Dunn [Harris, R. M., and Dunn, M. F. (2002) Biochemistry 41, 9982-9990] showed that when the novel amino acid, dihydroiso-L-tryptophan (DIT), reacts with the beta-site, the alpha-aminoacrylate Schiff base, E(A-A), is formed and the enzyme releases indoline. The indoline produced exits the enzyme via the tunnel out the open alpha-site. When the alpha-site ligand (ASL) alpha-D,L-glycerol 3-phosphate (GP) binds and closes the alpha-site, indoline generated in the DIT reaction is trapped for a short period of time as the quinonoid intermediate in rapid equilibrium with bound indoline and the E(A-A) intermediate before leaking out of the closed enzyme. In this work, we use the DIT reaction and a new, high-affinity, ASL, N-(4-trifluoromethoxybenzenesulfonyl)-2-amino-1-ethyl phosphate (F9), to explore the mechanism of ligand leakage from the closed enzyme. It was found that F9 binding to the alpha-site is significantly more effective than GP in trapping indoline in the DIT reaction; however, leakage of indoline from the enzyme into solution still occurs. It was also found that a combination of benzimidazole (BZI) and GP provided even more effective trapping than F9. The new experiments with F9 and the combination of BZI and GP provide evidence that the coincident binding of GP and BZI at the alpha-site exhibits a strong synergistic effect that greatly slows the leakage of indoline in the DIT reaction and enhances the trapping effect. This synergism functions to tightly close the alpha-site and sends an allosteric signal that stabilizes the closed structure of the beta-site. These studies also support a mechanism for the escape of indoline through the alpha-site that is limited by ASL dissociation.     

Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the beta-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium.

In an effort to understand the catalytic mechanism of the tryptophan synthase beta-subunit from Salmonella typhimurium, possible functional active site residues have been identified (on the basis of the 3-D crystal structure of the bienzyme complex) and targeted for analysis utilizing site-directed mutagenesis. The chromophoric properties of the pyridoxal 5'-phosphate cofactor provide a particularly convenient and sensitive spectral probe to directly investigate changes in catalytic events which occur upon modification of the beta-subunit. Substitution of Asp for Glu 109 in the beta-subunit was found to alter both the catalytic activity and the substrate specificity of the beta-reaction. Steady-state kinetic data reveal that the beta-reaction catalyzed by the beta E109D alpha 2 beta 2 mutant enzyme complex is reduced 27-fold compared to the wild-type enzyme. Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy shows that the mutation does not seriously affect the pre-steady-state reaction of the beta E109D mutant with L-serine to form the alpha-aminoacrylate intermediate, E(A-A). Binding of the alpha-subunit specific ligand, alpha-glycerol phosphate (GP) to the alpha 2 beta 2 complex exerts the same allosteric effects on the beta-subunit as observed with the wild-type enzyme. However, the pre-steady-state spectral changes for the reaction of indole with E(A-A) show that the formation of the L-tryptophan quinonoid, E(Q3), is drastically altered. Discrimination against E(Q3) formation is also observed for the binding of L-tryptophan to the mutant alpha 2 beta 2 complex in the reverse reaction. In contrast, substitution of Asp for Glu 109 increases the apparent affinity of the beta E109D alpha-aminoacrylate complex for the indole analogue indoline and results in the increased rate of synthesis of the amino acid product dihydroiso-L-tryptophan. Thus, the mutation affects the covalent bond forming addition reactions and the nucleophile specificity of the beta-reaction catalyzed by the bienzyme complex.     

Allosteric regulation of tryptophan synthase channeling: the internal aldimine probed by trans-3-indole-3'-acrylate binding

Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.     

Intermediate trapping via a conformational switch in the Na(+)-activated tryptophan synthase bienzyme complex

The tryptophan synthase bienzyme complex channels substrate indole between the alpha- and beta-sites via a 25 A long interconnecting tunnel. Channeling efficiency is dependent upon a conformational switch in alphabeta-dimeric units between open conformations of low activity to which substrates bind and closed conformations of high activity wherein substrates react. In experiments designed to gain a better understanding of the linkage between chemical steps and conformational transitions in the catalytic cycle, the novel amino acid dihydroiso-L-tryptophan (DIT) was used as an analogue of L-Trp. In the forward reaction (indoline + L-Ser) to synthesize DIT, the quinonoid species, E(Q)(indoline), is formed quickly, while in the reverse reaction (DIT cleavage), the accumulation of E(Q)(indoline) occurs very slowly. Nevertheless, when the alpha-site substrate analogue alpha-D,L-glycerol phosphate (GP) is bound, DIT cleavage was found to give a rapid formation and dissipation of E(Q)(indoline) followed by a very slow reappearance of E(Q)(indoline). This result led to the conclusion that the reaction of DIT proceeds quickly through the quinonoid state to give indoline and the alpha-aminoacrylate Schiff base, E(A-A), both in the absence and in the presence of GP. In the absence of GP the slow conversion of E(A-A) to pyruvate and ammonium ion limits the rate of accumulation of free indoline and therefore the rate of buildup of E(Q)(indoline). However, when GP is bound to the alpha-site, the indoline generated by DIT cleavage in the first turnover is trapped within the enzyme complex, shifting the equilibrium distribution strongly in favor of E(Q)(indoline) as a consequence of the high local concentration of sequestered indoline. This sequestering is the result of a switching of alphabeta-subunit pairs to a closed conformation when GP binds to the alpha-site and E(A-A) and/or E(Q)(indoline) is formed at the beta-site, thereby trapping indoline inside. The decay of the transiently formed E(Q)(indoline) occurs due to leakage of indoline from the closed system.     

Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex

Allosteric interactions regulate substrate channeling in Salmonella typhimurium tryptophan synthase. The channeling of indole between the alpha- and beta-sites via the interconnecting 25 A tunnel is regulated by allosteric signaling arising from binding of ligand to the alpha-site, and covalent reaction of l-Ser at the beta-site. This signaling switches the alpha- and beta-subunits between open conformations of low activity and closed conformations of high activity. Our objective is to synthesize and characterize new classes of alpha-site ligands (ASLs) that mimic the binding of substrates, 3-indole-d-glycerol 3'-phosphate (IGP) or d-glyceraldehyde 3-phosphate (G3P), for use in the investigation of alpha-site-beta-site interactions. The new synthesized IGP analogues contain an aryl group linked to an O-phosphoethanolamine moiety through amide, sulfonamide, or thiourea groups. The G3P analogue, thiophosphoglycolohydroxamate, contains a hydroxamic acid group linked to a thiophosphate moiety. Crystal structures of the internal aldimine complexed with G3P and with three of the new ASLs are presented. These structural and solution studies of the ASL complexes with the internal aldimine form of the enzyme establish the following. (1) ASL binding occurs with high specificity and relatively high affinities at the alpha-site. (2) Binding of the new ASLs slows the entry of indole analogues into the beta-site by blocking the tunnel opening at the alpha-site. (3) ASL binding stabilizes the closed conformations of the beta-subunit for the alpha-aminoacrylate and quinonoid forms of the enzyme. (4) The new ASLs exhibit allosteric properties that parallel the behaviors of IGP and G3P.     

Tryptophan synthase, an allosteric molecular factory

Tryptophan synthase (TrpS) is a pyridoxal phosphate-containing bifunctional enzyme that catalyzes the last two steps in the biosynthesis of L-tryptophan. Indole, an intermediate generated at the active site of the alpha-subunit is channeled via a 25 A long tunnel to the beta-active site where it reacts with an aminoacrylate intermediate derived from L-serine. The two reactions are kept in phase by allosteric interactions between the two subunits. The recent development of novel alpha-site ligands and alpha-reaction transition state analogs combined with kinetic and crystal structure analysis of Salmonella typhimurium tryptophan synthase has provided new insights into the allosteric regulation of substrate channeling, the reaction mechanisms of the alpha and beta active sites, and the influence of structural dynamics.     

Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex.

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction. However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., Brzovi?, P., Drewe, W.F., Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an "open" to a "closed" structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open to the closed form.     

The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[(2)H]indole

The bacterial tryptophan synthase alpha(2)beta(2) complex catalyzes the final reactions in the biosynthesis of L-tryptophan. Indole is produced at the active site of the alpha-subunit and is transferred through a 25-30 A tunnel to the beta-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner proposed a two-step nucleophilic addition-tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[(2)H]indole reacts with the aminoacrylate intermediate. We have now observed a KIE of 1.4-2.0 in the reaction of 3-[(2)H]indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an alpha-subunit ligand, disodium alpha-glycerophosphate (Na(2)GP), is present. Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-[(2)H]indole with tryptophan synthase preincubated with L-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the L-Trp external aldimine at 423 nm. The addition of Na(2)GP dramatically slows the rate of reaction of indole with the alpha-aminoacrylate intermediate. A primary KIE is not observed in the reaction of 3-[(2)H]indole with the aminoacrylate complex of tryptophan synthase in the presence of Na(2)GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions. The reaction of 2-methylindole does not show a KIE, either in the presence of Na(+) or Na(2)GP. These results support the previously proposed mechanism for the beta-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.     

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase was studied. The tryptophan synthase (EC 4.2.1.20) of Escherichia coli catalyzed beta-substitution reaction of L-serine into L-tryptophan and the amino acid racemase (EC 5.1.1.10) of Pseudomonas putida catalyzed the racemization of D-serine simultaneously in one reactor. Under optimal conditions established for L-tryptophan production, a large-scale production of L-tryptophan was carried out in a 200-liter reactor using intact cells of E. coli and P. putida. After 24 h of incubation with intermittent indole feeding, 110 g liter-1 of L-tryptophan was formed in molar yields of 91 and 100% for added DL-serine and indole, respectively. Continuous production of L-tryptophan was also carried out using immobilized cells of E. coli and P. putida. The maximum concentration of L-tryptophan formed was 5.2 g liter-1 (99% molar yield for indole), and the concentration decreased to 4.2 g liter-1 after continuous operation for 20 days.     

Mechanisms of monovalent cation action in enzyme catalysis: the tryptophan synthase alpha-, beta-, and alpha beta-reactions.

The alpha-subunit of the tryptophan synthase bienzyme complex catalyzes the formation of indole from the cleavage of 3-indolyl-D-glyceraldehyde 3'-phosphate, while the beta-subunit utilizes L-serine and the indole produced at the alpha-site to form tryptophan. The replacement reaction catalyzed by the beta-subunit requires pyridoxal 5'-phosphate (PLP) as a cofactor. The beta-reaction occurs in two stages: in stage I, the first substrate, L-Ser, reacts with the enzyme-bound PLP cofactor to form an equilibrating mixture of the L-Ser Schiff base, E(Aex1), and the alpha-aminoacrylate Schiff base intermediate, E(A-A); in stage II, this intermediate reacts with the second substrate, indole, to form tryptophan. Monovalent cations (MVCs) are effectors of these processes [Woehl, E., and Dunn, M. F. (1995) Biochemistry 34, 9466-9476]. Herein, detailed kinetic dissections of stage II are described in the absence and in the presence of MVCs. The analyses presented complement the results of the preceding paper [Woehl, E., and Dunn, M. F. (1999) Biochemistry 38, XXXX-XXXX], which examines stage I, and confirm that the chemical and conformational processes in stage I establish the presence of two slowly interconverting conformations of E(A-A) that exhibit different reactivities in stage II. The pattern of kinetic isotope effects on the overall activity of the beta-reaction shows an MVC-mediated change in rate-limiting steps. In the absence of MVCs, the reaction of E(A-A) with indole becomes the rate-limiting step. In the presence of Na+ or K+, the conversion of E(Aex1) to E(A-A) is rate limiting, whereas some third process not subject to an isotope effect becomes rate determining for the NH4+-activated enzyme. The combined results from the preceding paper and from this study define the MVC effects, both for the reaction catalyzed by the beta-subunit and for the allosteric communication between the alpha- and beta-sites. Partial reaction-coordinate free energy diagrams and simulation studies of MVC effects on the proposed mechanism of the beta-reaction are presented.     

Production L-tryptophan by Escherichia coli cells

Whole cells of Escherichia coli B 10 having high tryptophan synthetase activity were used directly as an enzyme source to produce L-tryptophan from indole and L- or D,L-serine. This strain is tryptophan auxotrophic, which is tryptophanase negative and, in addition, L- and D-serine deaminase negative under production conditions. To avoid inhibition of tryptophan synthetase by a high concentration of indole, nonaqueous organic solvents, Amberlite XAD-2 adsorbent, and nonionic detergents were used as reservoirs of indole in the reaction mixture for the production of L-tryptophan. As a result, different effects were observed on the production of L-tryptophan. Particularly, among the nonionic detergents, Triton X-100 was very efficient. Using Triton X-100 for production of L-tryptophan from indole and L- or D,L-serine by whole cells of Escherichia coli B 10, 14.14 g/100 mL and 14.2 g/100 mL of L-tryptophan were produced at 37 degrees C for 60 h.     

Site-specific mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium. Changing arginine 179 to leucine alters the reciprocal transmission of substrate-induced conformational changes between the alpha and beta 2 subunits

Arginine 179 of the alpha subunit of tryptophan synthase of Salmonella typhimurium was changed to leucine by site-directed mutagenesis. The mutant alpha subunit was expressed in S. typhimurium, purified and crystallized as the alpha 2 beta 2 complex, and characterized by kinetic studies under steady-state reaction conditions. The rate of cleavage of indole 3-glycerol phosphate (alpha reaction) is reduced by 60% in the mutant alpha 2 beta 2 complex, whereas the rate of L-tryptophan synthesis from indole and L-serine (beta reaction) is unchanged. Thus, arginine 179 is not obligatory for catalysis, for binding of indole 3-glycerol phosphate, or for interaction of the alpha and beta 2 subunits. However, changing arginine 179 to leucine does have striking effects on ligand-dependent properties of this multienzyme complex. Ligands of the alpha subunit (DL-alpha-glycerophosphate and indole 3-propanol phosphate) which strongly inhibit the beta reaction of the native alpha 2 beta 2 complex have a slight stimulatory effect on the beta reaction of the mutant alpha 2 beta 2 complex. Likewise, L-serine, a ligand of the beta subunit which produces a 5-fold reduction in the Km for the alpha ligand indole 3-glycerol phosphate in the native alpha 2 beta 2 complex, has no effect on the mutant alpha 2 beta 2 complex. These results suggest that arginine 179 of the alpha subunit plays a role in the reciprocal transmission of substrate-induced conformational changes which occur between native alpha and beta 2 subunits in the alpha 2 beta 2 complex.     

Allosteric effects acting over a distance of 20-25 A in the Escherichia coli tryptophan synthase bienzyme complex increase ligand affinity and cause redistribution of covalent intermediates

The reactions of L-histidine (L-His) and L-tryptophan (L-Trp) with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase are introduced as probes both of beta-subunit catalysis and of ligand-mediated alpha-beta allosteric interactions. Binding of DL-alpha-glycerol 3-phosphate (GP), an analogue of 3-indole-D-glycerol 3'-phosphate (IGP), to the alpha-catalytic site increases the affinity of alpha 2 beta 2 for L-His 4.5-fold and the affinity for L-Trp 17-fold and brings about a redistribution of beta-bound intermediates that favors the quinonoids derived from each amino acid. Inorganic phosphate (Pi) (presumably via binding to the alpha-catalytic site) influences the distribution of L-His intermediates as does GP. Previous binding studies [Heyn, M. P., & Weischet, W. O. (1975) Biochemistry 14, 2962-2968] indicate that when the phosphoryl group subsite of the alpha-catalytic site is occupied by GP or Pi, a high-affinity indole subsite is induced at the alpha-catalytic site. Interaction of benzimidazole (BZ), an analogue of indole, with this site also shifts the distribution of beta-bound L-His intermediates in favor of the L-His quinonoid. In the absence of Pi or GP, BZ interacts primarily at the beta-catalytic site and competes with L-His for the beta-subunit indole subsite. Since L-His and GP (or Pi) are substrate analogues and L-Trp is the physiological product, these allosteric effects likely take place with the natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta D305A mutant of tryptophan synthase shows strongly perturbed allosteric regulation and substrate specificity.

Substrate channeling in the tryptophan synthase bienzyme is regulated by allosteric interactions. Allosteric signals are transmitted via a scaffolding of structural elements that includes a monovalent cation-binding site and salt-bridging interactions between the side chains of betaAsp 305, betaArg 141, betaLys 167, and alphaAsp 56 that appear to modulate the interconversion between open and closed conformations. betaAsp 305 also interacts with the hydroxyl group of the substrate L-Ser in some structures. One possible functional role for betaAsp 305 is to ensure the allosteric transmission that triggers the switching of alphabeta-dimeric units between open and closed conformations of low and high activity. This work shows that substitution of betaAsp 305 with Ala (betaD305A) decreases the affinity of the beta-site for the substrate L-Ser, destabilizes the enzyme-bound alpha-aminoacrylate, E(A-A), and quinonoid species, E(Q), and changes the nucleophile specificity of the beta-reaction. The altered specificity provides a biosynthetic route for new L-amino acids derived from substrate analogues. betaD305A also shows an increased rate of formation of pyruvate upon reaction with L-Ser relative to the wild-type enzyme. The formation of pyruvate is strongly inhibited by the binding of benzimidazole to E(A-A). Upon reaction with L-Ser and in the presence of the alpha-site substrate analogue, alpha-glycerol phosphate, the Na(+) form of betaD305A undergoes inactivation via reaction of nascent alpha-aminoacrylate with bound PLP. This work establishes important roles for betaAsp 305 both in the conformational change between open and closed states that takes place at the beta-site during the formation of the E(A-A) and in substrate binding and recognition.     

Beta-elimination of indole from L-tryptophan catalyzed by bacterial tryptophan synthase: a comparison between reactions catalyzed by tryptophanase and tryptophan synthase

Although tryptophan synthase catalyzes a number of pyridoxal phosphate dependent beta-elimination and beta-replacement reactions that are also catalyzed by tryptophanase, a principal and puzzling difference between the two enzymes lies in the apparent inability of tryptophan synthase to catalyze beta-elimination of indole from L-tryptophan. We now demonstrate for the first time that the beta 2 subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli and from Salmonella typhimurium do catalyze a slow beta-elimination reaction with L-tryptophan to produce indole, pyruvate, and ammonia. The rate of the reaction is about 10-fold higher in the presence of the alpha subunit. The rate of indole production is increased about 4-fold when the aminoacrylate produced is converted to S-(hydroxyethyl)-L-cysteine by a coupled beta-replacement reaction with beta-mercaptoethanol. The rate of L-tryptophan cleavage is also increased when the indole produced is removed by extraction with toluene or by condensation with D-glyceraldehyde 3-phosphate to form indole-3-glycerol phosphate in a reaction catalyzed by the alpha subunit of tryptophan synthase. The amount of L-tryptophan cleavage is greatest in the presence of both beta-mercaptoethanol and D-glyceraldehyde 3-phosphate, which cause the removal of both products of cleavage. The cleavage reaction is not due to contaminating tryptophanase since the activity is not inhibited by (3R)-2,3-dihydro-L-tryptophan, a specific inhibitor of tryptophanase, but is inhibited by (3S)-2,3-dihydro-L-tryptophan, a specific inhibitor of tryptophan synthase. The cleavage reaction is also inhibited by D-tryptophan, the product of a slow racemization reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-serine binds to arginine-148 of the beta 2 subunit of Escherichia coli tryptophan synthase

Inactivation of the beta 2 subunit and of the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of one arginyl residue per beta monomer. The substrate L-serine protects the holo beta 2 subunit and the holo alpha 2 beta 2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by both the holo beta 2 subunit and the holo alpha 2 beta 2 complex indicate that L-serine and phenylglyoxal both bind to the same essential arginyl residue in the holo beta 2 subunit. The apo beta 2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by either pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo beta 2 subunit binds pyridoxal phosphate and the alpha subunit but cannot bind L-serine or L-tryptophan. We conclude that the alpha-carboxyl group of L-serine and not the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the beta 2 subunit. The specific arginyl residue in the beta 2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal has been identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains three other basic residues (lysine-137, arginine-141, and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148, and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium, and yeast supports a functional role for these three residues in anion binding. The location and role of the active-site arginyl residues in the beta 2 subunit and in two other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.     

Photoaffinity labeling of the indole sites on the Escherichia coli tryptophan synthase alpha-subunit

The alpha subunit of the Escherichia coli tryptophan synthase catalyzes the reversible aldolytic reaction: Indole-3-glycerol phosphate in equilibrium indole + glyceraldehyde 3-phosphate. The use of 5-azidoindole as a photoaffinity label has made the generation of a number of enzyme-substrate complexes possible, each with a given degree of saturation of the two postulated indole sites. When assayed in the reverse reaction (indole-3-glycerol phosphate synthesis), samples of alpha subunit treated at concentrations of 5-azidoindole less than or equal to 2 mM show a progressive 30-40% activation. A gradual inactivation occurs only in samples irradiated at concentrations in excess of 2 mM 5-azidoindole, and this inactivation is complete at 8-10 mM. A quantitatively similar activation occurs in the forward reaction (indole synthesis), however inactivation in this case is incomplete, with complexes treated at 8-12 mM 5-azidoindole retaining 30-40% relative activity in this reaction. When treated alpha subunits were assayed for their abilities to complement the beta 2-subunit in the reactions indole + L-serine leads to L-tryptophan + H2O and indole-3-glycerol phosphate + L-serine leads to L-tryptophan + glyceraldehyde 3-phosphate, quantitatively lesser amounts of activation followed by total inactivation are observed over a similar range of 5-azidoindole concentrations.     

The catalytic mechanism of tryptophan synthase from Escherichia coli. Kinetics of the reaction of indole with the enzyme--L-serine complexes

The mechanism by which indole condenses with L-serine in the active site of tryptophan synthase was studied by the stopped-flow technique. The single turnover occurs by rapid binding of indole to the pre-formed enzyme--L-serine complex, followed by C--C bond formation, reprotonation of the alpha carbon carbanion of L-tryptophan, and its final release. The effects of isotopic substitution at C-3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L-serine and L-tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme--L-serine complexes, leading to a branched pathway for the central condensation process. The rates of dehydration of L-serine and reprotonation of the carbanion of L-tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C-4' atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the alpha subunit on the reaction catalyzed by the beta 2 subunit.