Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Tryptophan Synthase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structures of a new class of allosteric effectors complexed to tryptophan synthase.

Tryptophan synthase is a bifunctional alpha(2)beta(2) complex catalyzing the last two steps of l-tryptophan biosynthesis. The natural substrates of the alpha-subunit indole- 3-glycerolphosphate and glyceraldehyde-3-phosphate, and the substrate analogs indole-3-propanolphosphate and dl-alpha-glycerol-3-phosphate are allosteric effectors of the beta-subunit activity. It has been shown recently, that the indole-3-acetyl amino acids indole-3-acetylglycine and indole-3-acetyl-l-aspartic acid are both alpha-subunit inhibitors and beta-subunit allosteric effectors, whereas indole-3-acetyl-l-valine is only an alpha-subunit inhibitor (Marabotti, A., Cozzini, P., and Mozzarelli, A. (2000) Biochim. Biophys. Acta 1476, 287-299). The crystal structures of tryptophan synthase complexed with indole-3-acetylglycine and indole-3-acetyl-l-aspartic acid show that both ligands bind to the active site such that the carboxylate moiety is positioned similarly as the phosphate group of the natural substrates. As a consequence, the residues of the alpha-active site that interact with the ligands are the same as observed in the indole 3-glycerolphosphate-enzyme complex. Ligand binding leads to closure of loop alphaL6 of the alpha-subunit, a key structural element of intersubunit communication. This is in keeping with the allosteric role played by these compounds. The structure of the enzyme complex with indole-3-acetyl-l-valine is quite different. Due to the hydrophobic lateral chain, this molecule adopts a new orientation in the alpha-active site. In this case, closure of loop alphaL6 is no longer observed, in agreement with its functioning only as an inhibitor of the alpha-subunit reaction.

Allosteric Site↗
PubMed1999

Cooperative fluctuations and subunit communication in tryptophan synthase.

Tryptophan synthase (TRPS), with linearly arrayed subunits alphabetabetaalpha, catalyzes the last two reactions in the biosynthesis of L-tryptophan. The two reactions take place in the respective alpha- and beta-subunits of the enzyme, and the intermediate product, indole, is transferred from the alpha- to the beta-site through a 25 A long hydrophobic tunnel. The occurrence of a unique ligand-mediated long-range cooperativity for substrate channeling, and a quest to understand the mechanism of allosteric control and coordination in metabolic cycles, have motivated many experimental studies on the structure and catalytic activity of the TRPS alpha2beta2 complex and its mutants. The dynamics of these complexes are analyzed here using a simple but rigorous theoretical approach, the Gaussian network model. Both wild-type and mutant structures, in the unliganded and various liganded forms, are considered. The substrate binding site in the beta-subunit is found to be closely coupled to a group of hinge residues (beta77-beta89 and beta376-beta379) near the beta-beta interface. These residues simultaneously control the anticorrelated motion of the two beta-subunits, and the opening or closing of the hydrophobic tunnel. The latter process is achieved by the large amplitude fluctuations of the so-called COMM domain in the same subunit. Intersubunit communications are strengthened in the presence of external aldimines bound to the beta-site. The motions of the COMM core residues are coordinated with those of the alpha-beta hinge residues beta174-beta179 on the interfacial helix betaH6 at the entrance of the hydrophobic tunnel. And the motions of betaH6 are coupled, via helix betaH1 and alphaL6, to those of the loop alphaL2 that includes the alpha-subunit catalytically active residue Asp60. Overall, our analysis sheds light on the molecular machinery underlying subunit communication, and identifies the residues playing a key role in the cooperative transmission of conformational motions across the two reaction sites.

Allosteric Regulation↗
PubMed1995

Kinetic characterization of channel impaired mutants of tryptophan synthase.

Tryptophan synthase, an alpha 2 beta 2 tetrameric complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. The solution of the three-dimensional structure of the enzyme from Salmonella typhimurium provided physical evidence for a 25-A hydrophobic tunnel which connects the alpha and beta active sites (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). Using rapid reaction kinetics, we have previously established that indole is indeed channeled and have identified three essential kinetic features which govern efficient channeling. In the current study we have probed the necessity of these features by using site-directed mutagenesis to alter these requirements. We now report the kinetic characterization of two mutants which contain substitutions to block or restrict the tunnel (beta C170F and beta C170W). Preliminary kinetic and structural evidence of a restricted tunnel in the beta C170W has been provided (Schlichting, I., Yang, X. W., Miles, E. W., Kim, A. Y., and Anderson, K. S. (1994) J. Biol. Chem. 269, 26591-26593). The rapid kinetic analysis of these mutant proteins shows that these mutations interfere with efficient channeling of the indole metabolite such that indole can be observed in single enzyme turnover of the physiologically relevant alpha beta reaction. In addition, the beta C170W mutant appears to be impaired in alpha beta intersubunit communication.

Binding Sites↗
PubMed1986

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)

Escherichia coli↗
PubMed2005

Conformational changes in the alpha-subunit coupled to binding of the beta 2-subunit of tryptophan synthase from Escherichia coli: crystal structure of the tryptophan synthase alpha-subunit alone.

When the tryptophan synthase alpha- and beta(2)-subunits combine to form the alpha(2)beta(2)-complex, the enzymatic activity of each subunit is stimulated by 1-2 orders of magnitude. To elucidate the structural basis of this mutual activation, it is necessary to determine the structures of the alpha- and beta-subunits alone and together with the alpha(2)beta(2)-complex. The crystal structures of the tryptophan synthase alpha(2)beta(2)-complex from Salmonella typhimurium (Stalpha(2)beta(2)-complex) have already been reported. However, the structures of the subunit alone from mesophiles have not yet been determined. The structure of the tryptophan synthase alpha-subunit alone from Escherichia coli (Ecalpha-subunit) was determined by an X-ray crystallographic analysis at 2.3 A, which is the first report on the subunits alone from the mesophiles. The biggest difference between the structures of the Ecalpha-subunit alone and the alpha-subunit in the Stalpha(2)beta(2)-complex (Stalpha-subunit) was as follows. Helix 2' in the Stalpha-subunit, including an active site residue (Asp60), was changed to a flexible loop in the Ecalpha-subunit alone. The conversion of the helix to a loop resulted in the collapse of the correct active site conformation. This region is also an important part for the mutual activation in the Stalpha(2)beta(2)-complex and interaction with the beta-subunit. These results suggest that the formation of helix 2'that is essential for the stimulation of the enzymatic activity of the alpha-subunit is constructed by the induced-fit mode involved in conformational changes upon interaction between the alpha- and beta-subunits. This also confirms the prediction of the conformational changes based on the thermodynamic analysis for the association between the alpha- and beta-subunits.

Amino Acid Sequence↗
PubMed2006

Evolution of multi-enzyme complexes: the case of tryptophan synthase.

The prototypical tryptophan synthase is a stable heterotetrameric alpha-betabeta-alpha complex. The constituting TrpA and TrpB1 subunits, which are encoded by neighboring genes in the trp operon, activate each other in a bi-directional manner. Recently, a novel class of TrpB2 proteins has been identified, whose members contain additional amino acids that might sterically prevent complex formation with TrpA. To test this hypothesis, we characterized the TrpA and TrpB proteins from Sulfolobus solfataricus. This hyperthermophilic archaeon does not contain a TrpB1 protein but instead contains two TrpB2 homologues that are encoded within (TrpB2i) and outside (TrpB2o) the trp operon. We find that TrpB2i and TrpA form a weak and transient complex during catalysis, with a uni-directional activation of TrpA by TrpB2i. In contrast, TrpB2o and TrpA do not form a detectable complex. These results suggest a model for the evolution of the tryptophan synthase in which TrpB2o, TrpB2i, and TrpB1 reflect the stepwise increase of TrpB affinity for TrpA and the refinement of functional subunit interaction, concomitant with the co-localization of the encoding genes in the trp operon.

Amino Acid Sequence↗
PubMed1991

Reciprocal communication between the lyase and synthase active sites of the tryptophan synthase bienzyme complex.

It is important to understand how the cleavage of indoleglycerol phosphate, which is catalyzed by the alpha subunits in the alpha 2 beta 2 bienzyme complex of tryptophan synthase, is modulated by the presence of L-serine in the beta subunits. Steady-state kinetic data, including the dependence of kcat on pH, allowed values to be assigned to each of the eight rate constants of the minimal catalytic mechanism. An ionizing group having an apparent pK value near 7.5 must be protonated for activity. The alpha active site ligands indolepropanol phosphate, glyceraldehyde 3-phosphate, and glycerol 3-phosphate increase both the affinity and the molar absorbance of L-serine and L-tryptophan bound to the beta active site. These effects prove that the alpha sites communicate with the beta sites over a distance of 30 A. 6-Nitroindole readily condenses with glyceraldehyde 3-phosphate, but not with L-serine. The turnover numbers for 6-nitroindoleglycerol phosphate and 6-nitroindole increased about 10-fold in both directions in the presence of L-serine bound to the beta 2 subunits. These data prove that the alpha and beta active sites communicate reciprocally and explain why the turnover number for the physiological reaction of indoleglycerol phosphate with L-serine greatly exceeds that of the cleavage reaction of indoleglycerol phosphate.

Binding Sites↗
PubMed1992

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)

Alanine↗
PubMed1997

Tryptophan synthase from Agrobacterium tumefaciens 8628: isolation and properties.

Tryptophan synthase was isolated from a highly virulent strain of Agrobacterium tumefaciens 8628 (octopine type). Separation of tryptophan synthase from thermolabile protease was accomplished using fractionation with polyethylene glycol-6000 followed by ion-exchange chromatography with a pH gradient. Molecular weights of alpha- and beta-subunits are 33 and 51 kD, respectively. The tryptophan synthase is stable at 60 degrees C because of heat-tolerance beta-subunits. After heating the activity of tryptophan synthase increased up to 20 times while temperature-labile proteases lost their activities. Reaction with antibodies showed the presence of four protein bands, one of which was coeluted with nucleic acids during ion-exchange chromatography. It is suggested that the basic tryptophan synthase is encoded by trp genes in a plasmid and its role is to provide the precursor with the prokaryotic pathway of indole-3-acetic acid biosynthesis, which determines the virulence of A. tumefaciens. There is perhaps a cooperation between iaaM, iaaH, and trp genes in the plasmid during plant cell transformation.

Antibodies, Bacterial↗
PubMed1993

Inhibition of tryptophan synthase by (1-fluorovinyl)glycine.

Tryptophan synthase (alpha 2 beta 2 complex) from Salmonella typhimurium catalyzes the formation of tryptophan from serine and indole. The enzyme is inactivated by (1-fluorovinyl)glycine. Concomitant with enzyme inactivation, the absorbance at 485 nm increases, indicating covalent modification of pyridoxal 5'-phosphate. It is proposed that inactivation involves elimination of HF to form an allene, which reacts with a nucleophile at the active site. The inactivation reaction involves an alpha,beta-elimination, as does the formation of tryptophan from indole and serine. The inactivation occurs with k(in) > 1.3 s-1, which is very close to k(cat) (6.4 s-1) for the formation of tryptophan from indole and serine. The inactive enzyme (alpha 2 beta 2) regains activity with k(off) = 0.005 min-1. Aminoacetone is formed during reaction, and pyridoxal 5'-phosphate is regenerated. Tryptophan synthase also catalyzes the dehydration of serine, or 3-fluoroalanine, to pyruvate in the absence of indole. This reaction involves an alpha,beta-elimination and the intermediate formation of an aminoacrylate adduct with pyridoxal 5'-phosphate, as does the formation of tryptophan. Pyruvate formation proceeds at less than 5% the rate of tryptophan formation. With [2-2H]serine an isotope effect (DVmax = 1.5) is observed. We propose that pyruvate formation is limited by the rate of hydration of the aminoacrylate intermediate and the rate of the abstraction of the serine alpha-hydrogen.

Acetone↗
PubMed2000

Biochemical analysis of a thermostable tryptophan synthase from a hyperthermophilic archaeon.

Pyridoxal 5'-phosphate-dependent tryptophan synthase catalyzes the last two reactions of tryptophan biosynthesis, and is comprised of two distinct subunits, alpha and beta. TktrpA and TktrpB, which encode the alpha subunit and beta subunit of tryptophan synthase from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, were independently expressed in Escherichia coli and their protein products were purified. Tryptophan synthase complex (Tk-TS complex), obtained by heat treatment of a mixture of the cell-free extracts containing each subunit, was also purified. Gel-filtration chromatography revealed that Tk-TrpA was a monomer (alpha), Tk-TrpB was a dimer (beta2), and Tk-TS complex was a tetramer (alpha2 beta2). The Tk-TS complex catalyzed the overall alphabeta reaction with a specific activity of 110 micromol Trp per micromol active site per min under its optimal conditions (80 degrees C, pH 8.5). Individual activity of the alpha and beta reactions of the Tk-TS complex were 8.5 micromol indole per micromol active site per min (70 degrees C, pH 7.0) and 119 micromol Trp per micromol active site per min (90 degrees C, pH 7.0), respectively. The low activity of the alpha reaction of the Tk-TS complex indicated that turnover of the beta reaction, namely the consumption of indole, was necessary for efficient progression of the alpha reaction. The alpha and beta reaction activities of independently purified Tk-TrpA and Tk-TrpB were 10-fold lower than the respective activities detected from the Tk-TS complex, indicating that during heat treatment, each subunit was necessary for the other to obtain a proper conformation for high enzyme activity. Tk-TrpA showed only trace activities at all temperatures examined (40-85 degrees C). Tk-TrpB also displayed low levels of activity at temperatures below 70 degrees C. However, Tk-TrpB activity increased at temperatures above 70 degrees C, and eventually at 100 degrees C, reached an equivalent level of activity with the beta reaction activity of Tk-TS complex. Taking into account the results of circular dichroism analyses of the three enzymes, a model is proposed which explains the relationship between structure and activity of the alpha and beta subunits with changes in temperature. This is the first report of an archaeal tryptophan synthase, and the first biochemical analysis of a thermostable tryptophan synthase at high temperature.

Amino Acid Sequence↗
PubMed1984

Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan: support for an indolenine intermediate in tryptophan metabolism.

We have examined the interaction of tryptophan synthase and tryptophanase with the tryptophan analogues oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Since these analogues have tetrahedral geometry at carbon 3 of the heterocyclic ring, they are structurally similar to the indolenine tautomer of L-tryptophan, a proposed intermediate in reactions of L-tryptophan. Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan are potent competitive inhibitors of both tryptophan synthase and tryptophanase, with KI values (3-17 microM) 10-100-fold lower than the corresponding Km or KI values for L-tryptophan. Addition of oxindolyl-L-alanine or 2,3-dihydro-L-tryptophan to solutions of the alpha 2 beta 2 complex of tryptophan synthase results in new absorption bands at 480 or 494 nm, respectively, which are ascribed to a quinonoid or alpha-carbanion intermediate. Spectrophotometric titration data give half-saturation values of 5 and 25 microM, which are comparable to the KI values obtained in kinetic experiments. Our finding that both enzymes catalyze incorporation of tritium from 3H2O into oxindolyl-L-alanine is evidence that both enzymes form alpha-carbanion intermediates with oxindolyl-L-alanine. These results support the proposal that the indolenine tautomer of L-tryptophan is an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase. In addition, we have found that oxindolyl-L-alanine reacts irreversibly with free pyridoxal phosphate to form a covalent adduct.

Alanine↗
PubMed1999

Mechanisms of monovalent cation action in enzyme catalysis: the first stage of the tryptophan synthase beta-reaction.

The tryptophan synthase bienzyme complex is activated and regulated by the allosteric action of monovalent cations (MVCs). The kinetic dissection of the first stage (stage I) in the beta-reaction of tryptophan synthase, the reaction of L-serine with pyridoxal 5'-phosphate at the beta-site to give the alpha-aminoacrylate Schiff base intermediate, E(A-A), is here examined in the absence and presence of MVCs. This analysis reveals which of the individual steps are greatly affected in stage I and how the ground states and transition states are affected by MVCs. Kinetic studies in combination with a detailed relaxation kinetic analysis to determine the specific rate constants for the conversion of the L-Ser external aldimine, E(Aex1), to E(A-A) show that the primary kinetic isotope effect for proton abstraction from Calpha of the E(Aex1) species changes from 4.0 +/- 0.4 in the absence of MVCs to a value of 5.9 +/- 0.5 in the presence of Na+, indicating that the nature of the transition state for this C-H scission is significantly perturbed by the MVC effect. The E(A-A) species was found to exist in two conformations with different activities, the ratio of which is affected by the presence of MVCs. It is shown that changes in the rate constants of stage I are important in establishing the ratio of active to inactive conformations of the E(A-A) species. Consequently, the MVC effect alters the relative energies of both the transition states and the ground states for selected steps in stage I of the pathway. Hence, interactions at the MVC site give rise to a fine-tuning of the covalent bonding interactions between active site residues and the reacting substrate during the conformational cycle of the bienzyme complex.

Binding Sites↗
PubMed1984

Reaction of phenylglyoxal and (p-hydroxyphenyl) glyoxal with arginines and cysteines in the alpha subunit of tryptophan synthase.

The alpha subunit of tryptophan synthase from Escherichia coli is inactivated by phenylglyoxal and by (p-hydroxyphenyl)glyoxal. The use of these chemical modification reagents to determine the role of arginyl residues in the alpha subunit of tryptophan synthase has been complicated by our finding that these reagents react with sulfhydryl groups of the alpha subunit, as well as with arginyl residues. Analyses of the data for incorporation of phenyl[2-14C]glyoxal, for inactivation, and for sulfhydryl modification in the presence and absence of indole-3-glycerol phosphate indicate that two sulfhydryl groups and one arginine are essential for the activity. Our finding that the substrate protects the single essential arginyl residue but not the two sulfhydryl groups is consistent with the observed kinetics of partial protection by substrate or by a substrate analogue, indole-3-propanol phosphate. In contrast to phenylglyoxal, (p-hydroxyphenyl)glyoxal modifies two to three sulhydryl groups that are not protected by indole-3-glycerol phosphate and modifies none of the arginyl residues that are modified by phenylglyoxal.

Arginine↗
PubMed1986

Production of a novel tryptophan analog, beta-1-indazole-L-alanine with tryptophan synthase of Escherichia coli.

The tryptophan synthase alpha 2 beta 2 complex from Escherichia coli has been found to catalyze the beta-replacement reaction of L-serine with indazole, an indole analog which has a nitrogen atom at the 2-position (pyrazole ring). The reaction product was isolated and identified as beta-indazolealanine by mass spectrometric, elemental and NMR analyses. Careful assignment of 1H- and 13C-signals with several NMR techniques revealed that the beta-carbon of the product alanine moiety was bound to the 1-N-position of the indazole ring. This is the first example of the beta-replacement reaction catalyzed by tryptophan synthase occurring at any other position than the 3-position of indole analogs.

Chemical Phenomena↗
PubMed1992

Characterization of tryptophan and coenzyme luminescence in tryptophan synthase from Salmonella typhimurium.

Tryptophan synthase from Salmonella typhimurium is a bifunctional alpha 2 beta 2 complex that catalyzes the formation of L-tryptophan. We have characterized over the temperature range from 160 to 293 K the fluorescence and phosphorescence properties of the single tryptophan present at position 177 of the beta-subunit and of the pyridoxal 5'-phosphate bound through a Schiff's base in the beta-active site. The comparison between the fluorescence of the pyridoxal phosphate bound either to the protein or to valine free in solution indicates substantial protection for the coenzyme against thermal quenching and a greater intensity of the ketoenamine tautomer band. Trp-177 is highly luminescent, and its proximity to the pyridoxal moiety leads to an over 50% quenching of its fluorescence with both reduced and native coenzyme. The Trp phosphorescence spectrum possesses a narrow, well-defined, 0-0 vibrational band centered at 418.5 nm, a wavelength that indicates strong polar interactions with neighboring charges. The observation of delayed fluorescence in the native complex implies that the excited triplet state is involved in a process of triplet-singlet energy transfer to the ketoenamine tautomer. The rate of energy transfer, heterogeneous in low-temperature glasses with rate constants of 2.26 and 0.07 s-1, becomes homogeneous in fluid solutions as the coenzyme tautomer interconversion is likely faster than the phosphorescence decay. In both apo- and holo-alpha 2 beta 2, the phosphorescence from Trp-177 is long-lived even at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed2002

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.

Allosteric Regulation↗
PubMed1975

The subunit structure of tryptophan synthase from Neurospora crassa.

Tryptophan synthase of Neurospora crassa was purified to electrophoretic homogeneity from the wild type strain 74A which had been derepressed by the presence of 0.5 mM indoleacrylic acid in the growth medium. The isolated material migrated as a single symmetrical peak in the ultracentrifuge with a sedimentation constant of 6.0 S. Gel filtration on Sephadex G-200 AND CONVENTIONAL SEDIMENTATION EQUILIBIRIUM YIELDED MOLECULAR WEIGHT ESTIMATES OF 151,000 PLUS AND MINUS 10,000 AND 149,000 PLUS AND MINUS 10,000, RESPECTIVELY. Treatment of the enzyme with sodium dodecyl sulfate followed by polyacrylamide gel electrophoresis gave a single band with a relative mobility suggesting a molecular weight of 76,000 plus and minus 2000. Aspartic acid was the only detectable NH2-terminal amino acid and experiments with carboxypeptides A and B revealed that the three amino acids, isoleucine, leucine, and phenylalanine, were released rapidly and in the order mentioned. These results are interpreted as indicating that the Neurospora enzyme is a homodimer.

Amino Acid Sequence↗