Detection and identification of intermediates in the reaction of L-serine with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the UV-visible absorption changes (300-550 nm) that occur in the spectrum of enzyme-bound pyridoxal 5'-phosphate during the reaction of L-serine with the alpha 2 beta 2 and beta 2 forms of Escherichia coli tryptophan synthase. In agreement with previous kinetic studies [Lane, A., & Kirschner, K. (1983) Eur. J. Biochem. 129, 561-570], the reaction with alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3). The RSSF data reveal that during tau 1, the internal aldimine, E(PLP), with lambda max = 412 nm (pH 7.8), undergoes rapid conversion to two transient species, one with lambda max congruent to 420 nm and one with lambda max congruent to 460 nm. These species decay in a biphasic process (1/tau 2, 1/tau 3) to a complicated final spectrum with lambda max congruent to 350 nm and with a broad envelope of absorbance extending out to approximately 525 nm. Analysis of the time-resolved spectra establishes that the spectral changes in tau 2 are nearly identical with the spectral changes in tau 3. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to increase the amount of the 420-nm transient and to decrease the amount of the species with lambda max congruent to 460 nm. These findings identify the serine Schiff base (the external aldimine) as the 420 nm absorbing, highly fluorescent transient; the species with lambda max congruent to 460 nm is the delocalized carbanion (quinoidal) species derived from abstraction of the alpha proton from the external aldimine. The reaction of L-serine with beta 2 consists of two relaxations (1/tau 1 beta greater than 1/tau 2 beta) and yields a quasi-stable species with lambda max = 420 nm, in good agreement with a previous report [Miles, E. W., Hatanaka, M., & Crawford, I. P. (1968) Biochemistry 7, 2742-2753]. Analysis of the RSSF spectra indicates that the same spectral change occurs in each phase of the reaction. The similarity of the spectral changes that occur in tau 2 and tau 3 of the alpha 2 beta 2 reaction is postulated to originate from the existence of two (slowly) interconverting forms of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Application of rapid-scanning, stopped-flow spectroscopy to the characterization of intermediates formed in the reactions of L- and D-tryptophan and beta-mercaptoethanol with Escherichia coli tryptophan synthase

The reactions of the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase with D- and L-Trp and the presteady-state reaction of L-Ser and beta-mercaptoethanol under different premixing conditions have been investigated by rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy. The reaction of alpha 2 beta 2 with L-Ser and beta-mercaptoethanol occurs in 3 detectable relaxations with rates similar to the 3 relaxations seen in the partial reaction with L-Ser and in the reaction with L-Ser and indole. The presteady-state phase of the reaction of beta-mercaptoethanol with the alpha-aminoacrylate intermediate is characterized by 2 relaxations. The RSSF spectra for this reaction show that the spectral changes that take place in these 2 phases are essentially identical. The L-Trp reaction is biphasic, and the spectral changes occurring in each phase of the reaction also are identical. The 2 new spectral bands formed (lambda max congruent to 420 nm and congruent to 476 nm) are assigned as the L-Trp external aldimine (Schiff's base) and L-Trp quinonoid intermediates, respectively. The reaction of D-Trp also is biphasic. Analysis of first and second derivatives of the RSSF spectral changes give evidence for the formation of spectral bands with lambda max of approximately 423 nm, approximately 450 nm, and approximately 478 nm. The positions and shapes of these bands suggest a D-Trp external aldimine structure (423 nm) and a quinonoidal species (450 and 478 nm). However, product studies do not support this latter assignment. The behavior of the D- and L-Trp reactions and the reaction of beta-mercaptoethanol with the alpha-aminoacrylate strongly indicate the pre-existence of 2 slowly equilibrating forms of the internal aldimine and of the alpha-aminoacrylate.     

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.     

pH dependence of tryptophan synthase catalytic mechanism: I. The first stage, the beta-elimination reaction

The pyridoxal 5'-phosphate-dependent beta-subunit of the tryptophan synthase alpha(2)beta(2) complex catalyzes the condensation of L-serine with indole to form L-tryptophan. The first stage of the reaction is a beta-elimination that involves a very fast interconversion of the internal aldimine in a highly fluorescent L-serine external aldimine that decays, via the alpha-carbon proton removal and beta-hydroxyl group release, to the alpha-aminoacrylate Schiff base. This reaction is influenced by protons, monovalent cations, and alpha-subunit ligands that modulate the distribution between open and closed conformations. In order to identify the ionizable residues that might assist catalysis, we have investigated the pH dependence of the rate of the external aldimine decay by rapid scanning UV-visible absorption and single wavelength fluorescence stopped flow. In the pH range 6-9, the reaction was found to be biphasic with the first phase (rate constants k(1)) accounting for more than 70% of the signal change. In the absence of monovalent cations or in the presence of sodium and potassium ions, the pH dependence of k(1) exhibits a bell shaped profile characterized by a pK(a1) of about 6 and a pK(a2) of about 9, whereas in the presence of cesium ions, the pH dependence exhibits a saturation profile characterized by a single pK(a) of 9. The presence of the allosteric effector indole acetylglycine increases the rate of reaction without altering the pH profile and pK(a) values. By combining structural information for the internal aldimine, the external aldimine, and the alpha-aminoacrylate with kinetic data on the wild type enzyme and beta-active site mutants, we have tentatively assigned pK(a1) to betaAsp-305 and pK(a2) to betaLys-87. The loss of pK(a1) in the presence of cesium ions might be due to a shift to lower values, caused by the selective stabilization of a closed form of the beta-subunit.     

Control of ionizable residues in the catalytic mechanism of tryptophan synthase from Salmonella typhimurium

The tryptophan synthase alpha2beta2 complex catalyzes the last two steps in the biosynthesis of l-tryptophan in bacteria, plants, and fungi, the conversion of indole-3-glycerol phosphate and l-serine to l-tryptophan, glyceraldehyde 3-phosphate, and water. The beta-subunit binds pyridoxal 5'-phosphate and catalyzes the beta-replacement reaction with serine and indole. Structural, spectral, and kinetic studies indicate that different monovalent cations stabilize the alternative enzyme conformations and equilibrium distribution of the internal, external, and alpha-aminoacrylate Schiff base. To improve our understanding of the role of monovalent cations, the pH dependence of steady-state and pre-steady-state kinetic parameters and primary kinetic deuterium isotope effects were measured in the presence of l-serine and [alpha-2H]-l-serine in the absence and presence of Na+, K+, and Cs+. For the interpretation of the data obtained in this study, it was necessary to re-interpret a number of results published previously. Overall, data suggest that the enzyme exists in two conformers that equilibrate slowly either in the absence of substrates and monovalent cations or in the presence of K+ or Cs+, whereas they equilibrate faster in the presence of Na+. The rate of interconversion of the conformers increases as a group on the enzyme with a pKa of approximately 8 becomes deprotonated. The pH dependence of deuterium isotope effects is suggestive of a mechanism in which a pH-dependent conformational change that closes the active site precedes the chemical steps, likely a result of formation of one or more salt bridges. As the pH increases, the reaction becomes more committed to proceed to products, which causes the deuterium isotope effect to decrease to a value of unity at high pH. The closure of the site is modulated by the different monovalent cations and is fastest in the presence of Na+, which exhibits the maximum isotope effect of 5.7 (likely the intrinsic effect) on V/Kserine, and slowest in the presence of Cs+, which exhibits the smallest isotope effect of approximately 1.5. The isotope effect on V, in all cases, indicates a contribution to rate limitation from steps in the second half of the reaction. Finally, in the presence of Na+, the steady-state isotope effect on V is greater than that on the pre-steady-state rate constant for decay of the external Schiff base, suggesting that the rate of conversion of the two conformers of the internal aldimine contributes to the pre-steady-state rate, but not the steady-state rate because the high serine concentration traps the enzyme in the active E-serine complex before it can decay to the less active form.     

Reaction of serine-glyoxylate aminotransferase with the alternative substrate ketomalonate indicates rate-limiting protonation of a quinonoid intermediate

Serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the interconversion of L-serine and glyoxylate to hydroxypyruvate and glycine. The primary deuterium isotope effect using L-serine 2-D is one on (V/K)serine and V in the steady state. Pre-steady-state experiments also indicate that there is no primary deuterium isotope effect with L-serine 2-D. The results suggest there is no rate limitation by abstraction of the alpha proton of L-serine in the SGAT reaction. In the steady-state a solvent deuterium isotope effect of about 2 was measured on (V/K)L-serine and (V/K)ketomalonate and about 5.5 on V. Similar solvent isotope effects were observed in the pre-steady-state for the natural substrates and the alternative substrate ketomalonate. In the pre-steady-state, no reaction intermediates typical of PLP enzymes were observed with the substrates L-serine, glyoxylate, and hydroxypyruvate. The data suggest that breakdown and formation of the ketimine intermediate is the primary rate-limiting step with the natural substrates. In contrast, using the alternative substrate ketomalonate, pre-steady-state experiments display the transient formation of a 490 nm absorbing species typical of a quinonoid intermediate. The solvent isotope effect results also suggest that with ketomalonate as substrate protonation at C(alpha) is the slowest step in the SGAT reaction. This is the first report of a rate-limiting protonation of a quinonoid at C(alpha) of the external Schiff base in an aminotransferase reaction.     

L-serine analogues form Schiff base and quinonoidal intermediates with Escherichia coli tryptophan synthase

Substrate analogues of L-serine have been found that react with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase. Upon reaction with alpha 2 beta 2, the analogues glycine, L-histidine, L-alanine, and D-histidine form chemical intermediates derived from reaction with enzyme-bound pyridoxal 5'-phosphate with characteristic UV-visible spectral bands. The spectra of the products of the glycine, L-histidine, and L-alanine reactions with alpha 2 beta 2 contain contributions from the external aldimine, the quinonoid species, and other intermediates along the catalytic pathway. Just as previously reported for the reaction of L-serine with beta 2 [Goldberg, M. E., York, S., & Stryer, L. (1968) Biochemistry 7, 3662-3667], the reactions of glycine, L-histidine, and L-alanine with the beta 2 form of tryptophan synthase yield spectra with no contributions from catalytic intermediates beyond the external aldimine. The kinetics of intermediate formation and comparisons of the time courses for the exchange of alpha-1H for solvent 2H catalyzed by alpha 2 beta 2 or beta 2 were found to be consistent with these assignments. Intermediates further along the tryptophan synthase catalytic pathway are stabilized to a greater degree in the alpha 2 beta 2 complex than in the beta 2 species alone. This observation strongly suggests that the association of alpha and beta subunits to form the native alpha 2 beta 2 species lowers the activation energies for the interconversion of the external aldimine with chemical species further along the catalytic path.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The reaction of yeast cystathionine beta-synthase is rate-limited by the conversion of aminoacrylate to cystathionine

Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine. The second forward half-reaction of aminoacrylate with L-homocysteine is rapid. No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine. We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase.     

Guanidine hydrochloride exerts dual effects on the tryptophan synthase alpha 2 beta 2 complex as a cation activator and as a modulator of the active site conformation.

To characterize the conformational transitions that regulate the activity and specificity of the tryptophan synthase alpha 2 beta 2 complex, we have determined some effects of low concentrations of guanidine hydrochloride (GuHCl) and of urea on functional properties. We report the novel finding that GuHCl at low concentrations (0. 02-0.08 M) is a cation activator of the tryptophan synthase alpha 2 beta 2 complex. Molecular modeling studies show that GuH+ could bind at a previously identified cation binding site in the tryptophan synthase beta subunit. Addition of increasing concentrations of GuHCl has strikingly different effects on the rates of different reactions with L-serine or beta-chloro-L-alanine in the presence or absence of indole. Spectroscopic studies demonstrate that GuHCl alters the equilibrium distribution of pyridoxal 5'-phosphate intermediates formed in reactions at the active site of the beta subunit. Data analysis shows that GuHCl binds preferentially with the conformer of the enzyme that predominates when the aldimine of L-serine is formed and shifts the equilibrium in favor of this conformer. These results provide evidence that GuHCl exerts dual effects on tryptophan synthase as a cation, stimulating activity, and as a chaotropic agent, altering the distribution of conformational states that exhibit different reaction specificities. Our finding that the nonionic urea stabilizes the aldimine of L-serine in the presence, but not in the absence, of NaCl shows that cation binding plays an important role in the conformational transitions that regulate activity and the transmission of allosteric signals between the alpha and beta sites.     

On the mechanism of action of Escherichia coli tryptophan synthase. Steady-state investigations.

Tryptophan synthase from Escherichia coli (L-serine hydro-lyase (adding indole), EC 4.2.1.20) synthesizes L-trypotophan from indoleglycerol phosphate and L-serine, releasing glyceraldehyde 3-phosphate, or from indole and L-serine. The latter reaction (B reaction), catalyzed either by the beta2 species or by the (alpha2 beta2) complex, has been studied by steady-state methods. A sequential mechanism is indicated. Inhibition experiments with the substrate analogue benzimidazole were carried out in order to distinguish between random and ordered mechanisms. The results are compatible with a random sequential mechanism. The dissociation constants of the enzyme-substrate complexes are evaluated. When catalyzed by the tetrameric complex (alpha2 beta2) the B reaction is inhibited by higher concentrations of the substrate indole. This inhibition does not follow the usual substrate inhibition pattern. The question whether the binding of indole to the alpha-subunit exerts an inhibitory effect on the beta2 species, possibly by reversing the activation by the alpha subunit of the beta2 species, is discussed.     

Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism

Tryptophan synthase, an alpha 2 beta 2 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. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

Energetic adaptation of ligand binding to subunit structure of tryptophan synthase from Escherichia coli

The binding of indole and L-serine to the isolated alpha and beta 2 subunits and the native alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli was investigated by direct microcalorimetry to reveal the energetic adaptation of ligand binding to the subunit structure of a multienzyme complex. In contrast to the general finding that negative heat capacity changes are associated with ligand binding to proteins, complex formation of indole and the alpha subunit involves a small positive change in heat capacity. This unusual result was considered as being indicative of a loosening of the protein structure. Such an interpretation is in good agreement with results of chemical accessibility studies (Freedberg & Hardman, 1971). Whereas the thermodynamic parameters of indole binding are not influenced by the subunit interaction, the large negative change in heat capacity of -6.5 kJ/(K X mol of beta 2) measured for the binding of L-serine to the isolated beta 2 subunit disappears completely when serine interacts with the tetrameric complex. These data demonstrate that the energy transduction pattern and therefore the functional roles of the substrates indole and L-serine vary strongly with the subunit structure of tryptophan synthase.     

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.     

The accessibility of the active site and conformation states of the beta 2 subunit of tryptophan synthase studied by fluorescence quenching

The rate of quenching of the fluorescence of pyridoxal 5'-phosphate in the active site of the beta 2 subunit of tryptophan synthase from Escherichia coli was measured to estimate the accessibility of the coenzyme to the small molecules iodide and acrylamide. The alpha subunit and the substrate L-serine substantially reduced the quenching rate. For iodide, the order of decreasing quenching was: Schiff's base of N alpha-acetyl-lysine with pyridoxal 5'-phosphate greater than holo beta 2 subunit greater than holo alpha 2 beta 2 complex approximately equal to holo beta 2 subunit + L-serine greater than holo alpha 2 beta 2 complex + L-serine. The coenzyme in the beta 2 subunit is apparently freely accessible to both iodide and acrylamide (kappa approximately equal to 2 X 10(9) M-1 s-1), but the alpha subunit and L-serine decrease the rate by factors of 2-5. Quenching of the fluorescence of the single tryptophan residue of the beta 2 subunit revealed that the apo and holo forms exist in different states, whereas the alpha subunit stabilizes a third conformation. As the alpha subunit binds to the beta 2 subunit, the tryptophan residue, which is within 2.2 nm of the active site of the beta 2 subunit, probably rotates with respect to the plane of the ring of the coenzyme, such that fluorescence energy transfer from tryptophan to pyridoxal phosphate is greatly reduced. The alpha subunit strongly protects the active-site ligand indole propanol phosphate from quenching with acrylamide, consistent with the active site being deep in a cleft in the protein. Iodide induces dissociation of the holo alpha 2 beta 2 complex [E. W. Miles & M. Moriguchi (1977) J. Biol. Chem. 252, 6594-6599]. The effect of iodide on the fluorescence properties of holo alpha 2 beta 2 complex allows us to estimate an upper limit for the dissociation constant for the alpha 2 beta 2 complex of 10(-8) M, in the absence of iodide.     

BetaQ114N and betaT110V mutations reveal a critically important role of the substrate alpha-carboxylate site in the reaction specificity of tryptophan synthase

In the PLP-requiring alpha2beta2 tryptophan synthase complex, recognition of the substrate l-Ser at the beta-site includes a loop structure (residues beta110-115) extensively H-bonded to the substrate alpha-carboxylate. To investigate the relationship of this subsite to catalytic function and to the regulation of substrate channeling, two loop mutants were constructed: betaThr110 --> Val, and betaGln114 --> Asn. The betaT110V mutation greatly impairs both catalytic activity in the beta-reaction, and allosteric communication between the alpha- and beta-sites. The crystal structure of the betaT110V mutant shows that the modified l-Ser carboxylate subsite has altered protein interactions that impair beta-site catalysis and the communication of allosteric signals between the alpha- and beta-sites. Purified betaQ114N consists of two species of mutant protein, one with a reddish color (lambdamax = 506 nm). The reddish species is unable to react with l-Ser. The second betaQ114N species displays significant catalytic activities; however, intermediates obtained on reaction with substrate l-Ser and substrate analogues exhibit perturbed UV/vis absorption spectra. Incubation with l-Ser results in the formation of an inactive species during the first 15 min with lambdamax approximately 320 nm, followed by a slower conversion over 24 h to the species with lambdamax = 506 nm. The 320 and 506 nm species originate from conversion of the alpha-aminoacrylate external aldimine to the internal aldimine and alpha-aminoacrylate, followed by the nucleophilic attack of alpha-aminoacrylate on C-4' of the internal aldimine to give a covalent adduct with PLP. Subsequent treatment with sodium hydroxide releases a modified coenzyme consisting of a vinylglyoxylic acid moiety linked through C-4' to the 4-position of the pyridine ring. We conclude that the shortening of the side chain accompanying the replacement of beta114-Gln by Asn relaxes the steric constraints that prevent this reaction in the wild-type enzyme. This study reveals a new layer of structure-function interactions essential for reaction specificity in tryptophan synthase.     

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)     

Thermodynamics of the reactions catalyzed by the multifunctional enzyme complex tryptophan synthase

The intrinsic enthalpy changes (corrected for hydration of D-glyceraldehyde 3-phosphate) for the reactions catalyzed by the alpha and beta 2 subunits of tryptophan synthase from Escherichia coli have been determined calorimetrically. Cleavage of indoleglycerol phosphate (alpha reaction) was found to be associated with a delta H value of 54.0 +/- 2.5 kJ mol-1, while condensation of indole with L-serine (beta reaction) involved -80.3 +/- 4.6 kJ mol-1'. By direct determination of the enthalpy concomitant with the overall synthesis of tryptophan from indoleglycerol phosphate and L-serine an enthalpy value of -13.4 +/- 5.6 kJ mol-1 was observed. In view of the uncertainties of the literature data used for calculation of the hydration contribution, the agreement between the directly measured delta H value of the overall reaction and the sum of the enthalpies of the alpha and beta reactions is fair. Deamination of L-serine, a side reaction catalyzed preferentially by the isolated beta 2 pyridoxal 5'-phosphate2 subunit, was shown to be associated with an enthalpy change of -7.3 +/- 0.4 kJ mol-1.     

The tryptophan synthase bienzyme complex transfers indole between the alpha- and beta-sites via a 25-30 A long tunnel

The bacterial tryptophan synthase bienzyme complexes (with subunit composition alpha 2 beta 2) catalyze the last two steps in the biosynthesis of L-tryptophan. For L-tryptophan synthesis, indole, the common metabolite, must be transferred by some mechanism from the alpha-catalytic site to the beta-catalytic site. The X-ray structure of the Salmonella typhimurium tryptophan synthase shows the catalytic sites of each alpha-beta subunit pair are connected by a 25-30 A long tunnel [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Since the S. typhimurium and Escherichia coli enzymes have nearly identical sequences, the E. coli enzyme must have a similar tunnel. Herein, rapid kinetic studies in combination with chemical probes that signal the bond formation step between indole (or nucleophilic indole analogues) and the alpha-aminoacrylate Schiff base intermediate, E(A-A), bound to the beta-site are used to investigate tunnel function in the E. coli enzyme. If the tunnel is the physical conduit for the transfer of indole from the alpha-site to the beta-site, then ligands that block the tunnel should also inhibit the rate at which indole and indole analogues from external solution react with E(A-A). We have found that when D,L-alpha-glycerol 3-phosphate (GP) is bound to the alpha-site, the rate of reaction of indole and nucleophilic indole analogues with E(A-A) is strongly inhibited. These compounds appear to gain access to the beta-site via the alpha-site and the tunnel, and this access is blocked by the binding of GP to the alpha-site. However, when small nucleophiles such as hydroxylamine, hydrazine, or N-methylhydroxylamine are substituted for indole, the rate of quinonoid formation is only slightly affected by the binding of GP. Furthermore, the reactions of L-serine and L-tryptophan with alpha 2 beta 2 show only small rate effects due to the binding of GP. From these experiments, we draw the following conclusions: (1) L-Serine and L-tryptophan gain access to the beta-site of alpha 2 beta 2 directly from solution. (2) The small effects of GP on the rates of the L-serine and L-tryptophan reactions are due to GP-mediated allosteric interactions between the alpha- and beta-sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the allosteric anion-binding site of O-acetylserine sulfhydrylase.

A new crystal structure of the A-isozyme of O-acetylserine sulfhydrylase-A (OASS) with chloride bound to an allosteric site located at the dimer interface has recently been determined [Burkhard, P., Tai, C.-H., Jansonius, J. N., and Cook, P. F. (2000) J. Mol. Biol. 303, 279-286]. Data have been obtained from steady state and presteady-state kinetic studies and from UV-visible spectral studies to characterize the allosteric anion-binding site. Data obtained with chloride and sulfate as inhibitors indicate the following: (i) chloride and sulfate prevent the formation of the external aldimines with L-cysteine or L-serine; (ii) chloride and sulfate increase the external aldimine dissociation constants for O-acetyl-L-serine, L-methionine, and 5-oxo-L-norleucine; (iii) chloride and sulfate bind to the allosteric site in the internal aldimine and alpha-aminoacrylate external aldimine forms of OASS; (iv) sulfate also binds to the active site. Sulfide behaves in a manner identical to chloride and sulfate in preventing the formation of the L-serine external aldimine. The binding of chloride to the allosteric site is pH independent over the pH range 7-9, suggesting no ionizable enzyme side chains ionize over this pH range. Inhibition by sulfide is potent (K(d) is 25 microM at pH 8) suggesting that SH(-) is the physiologic inhibitory species.