Mechanism of the physiological reaction catalyzed by tryptophan synthase from Escherichia coli

The physiological synthesis of L-tryptophan from indoleglycerol phosphate and L-serine catalyzed by the alpha 2 beta 2 bienzyme complex of tryptophan synthase requires spatial and dynamic cooperation between the two distant alpha and beta active sites. The carbanion of the adduct of L-tryptophan to pyridoxal phosphate accumulated during the steady state of the catalyzed reaction. Moreover, it was formed transiently and without a lag in single turnovers, and glyceraldehyde 3-phosphate was released only after formation of the carbanion. These and further data prove first that the affinity for indoleglycerol phosphate and its cleavage to indole in the alpha subunit are enhanced substantially by aminoacrylate bound to the beta subunit. This indirect activation explains why the turnover number of the physiological reaction is larger than that of the indoleglycerol phosphate cleavage reaction. Second, reprotonation of nascent tryptophan carbanion is rate limiting for overall tryptophan synthesis. Third, most of the indole generated in the active site of the alpha subunit is transferred directly to the active site of the beta subunit and only insignificant amounts pass through the solvent. Comparison of the single turnover rate constants with the known elementary rate constants of the partial reactions catalyzed by the alpha and beta active sites suggests that the cleavage reaction rather than the transfer of indole or its condensation with aminoacrylate is rate limiting for the formation of nascent tryptophan.     

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.     

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)     

The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching.

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1 at 23 degrees C. The rate does not depend on ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 +/- 0.3) . 10(5) dm3 . mol-1 . s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in alpha-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 . 10(5), 0.56 . 10(5) and 0.11 . 10(5) dm3 . mol-1 . s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues.     

Characterization of the reaction of L-serine and indole with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

The pre-steady-state reaction of indole and L-serine with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase has been investigated under different premixing conditions with rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy for the spectral range 300-550 nm. When alpha 2 beta 2 was mixed with indole and L-serine, the reaction of 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) with rate constants identical with the three relaxations seen in the partial reaction with L-serine [Drewe, W.F., Jr., & Dunn, M.F. (1985) Biochemistry 24, 3977-3987]. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to be similar to the effects observed in the reaction with serine only. The observed spectral changes and isotope effects indicate that the aldimine of L-serine and PLP and the first quinoid derived from this external aldimine are transient species that accumulate during tau 1. Conversion of these intermediates to the alpha-aminoacrylate Schiff base during tau 2 and tau 3 limits the rate of formation of the second quinoidal species (lambda max 476 nm) generated via C-C bond formation between indole and the alpha-aminoacrylate intermediate. The pre-steady-state reaction of the alpha 2 beta 2-serine mixture with indole is comprised of four relaxations (1/tau 1* greater than 1/tau 2* greater than 1/tau 3* greater than 1/tau 4*).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Structural basis for the impaired channeling and allosteric inter-subunit communication in the beta A169L/beta C170W mutant of tryptophan synthase

We determined the 2.25 A resolution crystal structure of the betaA169L/betaC170W mutant form of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium complexed with the alpha-active site substrate analogue 5-fluoro-indole-propanol-phosphate to identify the structural basis for the changed kinetic properties of the mutant (Anderson, K. S., Kim, A. Y., Quillen, J. M., Sayers, E., Yang, X. J., and Miles, E. W. (1995) J. Biol. Chem. 270, 29936-29944). Comparison with the wild-type enzyme showed that the betaTrp(170) side chain occludes the tunnel connecting the alpha- and beta-active sites, explaining the accumulation of the intermediate indole during a single enzyme turnover. To prevent a steric clash between betaLeu(169) and betaGly(135), located in the beta-sheet of the COMM (communication) domain (betaGly(102)-betaGly(189)), the latter reorganizes. The changed COMM domain conformation results in a loss of the hydrogen bonding networks between the alpha- and beta-active sites, explaining the poor activation of the alpha-reaction upon formation of the aminoacrylate complex at the beta-active site. The 100-fold reduced affinity for serine seems to result from a movement of betaAsp(305) away from the beta-active site so that it cannot interact with the hydroxyl group of a pyridoxal phosphate-bound serine. The proposed structural dissection of the effects of each single mutation in the betaA169L/betaC170W mutant would explain the very different kinetics of this mutant and betaC170F.     

A novel tryptophan synthase beta-subunit from the hyperthermophile Thermotoga maritima. Quaternary structure, steady-state kinetics, and putative physiological role.

Tryptophan synthase catalyzes the last two steps in the biosynthesis of the amino acid tryptophan. The enzyme is an alpha beta beta alpha complex in mesophilic microorganisms. The alpha-subunit (TrpA) catalyzes the cleavage of indoleglycerol phosphate to glyceraldehyde 3-phosphate and indole, which is channeled to the active site of the associated beta-subunit (TrpB1), where it reacts with serine to yield tryptophan. The TrpA and TrpB1 proteins are encoded by the adjacent trpA and trpB1 genes in the trp operon. The genomes of many hyperthermophilic microorganisms, however, contain an additional trpB2 gene located outside of the trp operon. To reveal the properties and potential physiological role of TrpB2, the trpA, trpB1, and trpB2 genes of Thermotoga maritima were expressed heterologously in Escherichia coli, and the resulting proteins were purified and characterized. TrpA and TrpB1 form the familiar alpha beta beta alpha complex, in which the two different subunits strongly activate each other. In contrast, TrpB2 forms a beta(2)-homodimer that has a high catalytic efficiency k(cat)/K(m)(indole) because of a very low K(m)(indole) but does not bind to TrpA. These results suggest that TrpB2 acts as an indole rescue protein, which prevents the escape of this costly hydrophobic metabolite from the cell at the high growth temperatures of hyperthermophiles.     

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.     

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.     

The use of 6-(difluoromethyl)indole to study the activation of indole by tryptophan synthase

6-(Difluoromethyl)indole has been characterized and developed as a probe for the turnover of indole by the bifunctional enzyme, tryptophan synthase (alpha 2 beta 2). The neutral form of the indolyl species undergoes a slow and spontaneous hydrolysis to produce 6-formylindole with a rate constant (k1) of 0.0089 +/- 0.0001 min-1. The overall rate is independent of pH in the range of 3.5-10.5. Above pH 10.5, the observed rate increases are due to the high reactivity of the anionic form of the indole; deprotonation at N-1 accelerates hydrolysis by 10(4)-fold (k2, 97 +/- 2 min-1). The magnitude of this effect provides a technique for detecting the formation or stabilization of the anionic form of indole. 6-(Difluoromethyl)indole is recognized and processed by the beta subunit of tryptophan synthase. Selective inactivation of the beta subunit prevents enzymatic processing of 6-(difluoromethyl)indole. Chromatographic isolation and mass spectral analysis has identified 6-(difluoromethyl)tryptophan as the sole turnover product of the indolyl substrate. The lack of enzyme-promoted dehalogenation does not exclude the formation of an indole anion during turnover but rather the data suggest that rapid carbon-carbon bond formation (greater than 5300 min-1) prevents the accumulation of this anion.     

Conversion of Indolylglycerol Phosphate into Tryptophan by Extracts of Pisum

Dialyzed extracts from sterile and nonsterile pea seedlings perform the synthesis of tryptophan from indole-3-glycerol-1-phosphate (IGP) and L-serine. Compared with the analogous indole methabolism, there are similar dependences with regard to pH and to serine and pyridoxal-5-phosphate requirements, but the IGP conversion rate only amounts to one third of that measured for indole. Indole-3-glycerol is not metabolized by both tissues and homogenates.     

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.     

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.     

Kinetics of appearance of an early immunoreactive species during the refolding of acid-denatured Escherichia coli tryptophan synthase beta 2 subunit

A reversible acid-denaturation process of the beta 2 subunit of Escherichia coli tryptophan synthase has been set up. The acid-denatured state has been physically characterized: though not in a random-coiled conformation, it is extensively denatured. The renaturation of this denatured state of beta 2 has been observed in a stopped-flow system, in the presence of a monoclonal antibody directed against native beta 2. It is shown that the association occurs very early in the folding of beta 2. The association rate constants of the antibody with the immunoreactive folding intermediate and with native beta 2 are the same (3 X 10(5) M-1.s-1). But at high antibody concentrations the formation of the antigen/antibody complex is rate limited by a rapid (5.4 X 10(-2) s-1) isomerization of refolding beta chains. This isomerization appears to reflect the formation of at least part of the epitope recognized by the antibody during the folding of beta 2. Further conformational adjustments occurring later in the folding pathway would then allow the ultimate structuring of the epitope.     

Reaction of indole and analogues with amino acid complexes of Escherichia coli tryptophan indole-lyase: detection of a new reaction intermediate by rapid-scanning stopped-flow spectrophotometry

The effects of indole and analogues on the reaction of Escherichia coli tryptophan indole-lyase (tryptophanase) with amino acid substrates and quasisubstrates have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry. Indole binds rapidly (within the dead time of the stopped-flow instrument) to both the external aldimine and quinonoid complexes with L-alanine, and the absorbance of the quinonoid intermediate decreases in a subsequent slow relaxation. Indoline binds preferentially to the external aldimine complex with L-alanine, while benzimidazole binds selectively to the quinonoid complex of L-alanine. Indole and indoline do not significantly affect the spectrum of the quinonoid intermediates formed in the reaction of the enzyme with S-alkyl-L-cysteines, but benzimidazole causes a rapid decrease in the quinonoid peak at 512 nm and the appearance of a new peak at 345 nm. Benzimidazole also causes a rapid decrease in the quinonoid peak at 505 nm formed in the reaction with L-tryptophan and the appearance of a new absorbance peak at 345 nm. Furthermore, addition of benzimidazole to solutions of enzyme, potassium pyruvate, and ammonium chloride results in the formation of a similar absorption peak at 340 nm. This complex reacts rapidly with indole to form a quinonoid intermediate very similar to that formed from L-tryptophan. This new intermediate is formed faster than catalytic turnover (kcat = 6.8 s-1) and may be an alpha-aminoacrylate intermediate bound as a gem-diamine.     

Substrate-induced changes in the ammonia channel for imidazole glycerol phosphate synthase

IGP synthase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the unusual nucleotide, N(1)-[(5'-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to form 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR) and imidazole glycerol phosphate (IGP). A common feature of all glutamine amidotransferases is the upregulation of glutamine hydrolysis in the presence of an acceptor substrate. A refined assay system was developed to establish that Saccharomyces cerevisae IGP synthase shows a 4900-fold stimulation of glutaminase in the presence of the substrate acceptor PRFAR. The structure and function of IGP synthase acceptor substrate binding site were probed with competitive inhibitors that are nucleotide substrate and product analogues. In addition, these analogues were also used to establish that the normal steady-state turnover cycle involves a random sequential mechanism. Upregulation of the glutaminase active site occurs when these competitive inhibitors bind in the nucleotide site over 30 A away. One of the key structural features of IGP synthase is that the transfer of ammonia from the glutaminase site occurs through the (beta/alpha)(8) core of the protein. Upon the basis of the recent substrate-occupied structure for yeast IGP synthase (1), kinetic investigations of site-directed mutants revealed that a conserved K258 residue is key to productive binding and the overall stoichiometry of the reaction. The binding of the ribulosyl phosphate portion of the substrate PRFAR appears to be transduced through reorientation of K258 resulting in a conformational switch at the base of the (beta/alpha)(8) core that enables the passage of ammonia through the core of the protein. The overall analysis also leads to further discussion of how the residues that cover the opening of the (beta/alpha)(8) in the closed state may assist the channeling of ammonia at the interface of the two functional domains in the open state.     

Crystal structure of wild-type tryptophan synthase complexed with the natural substrate indole-3-glycerol phosphate

We used freeze trapping to stabilize the Michaelis complex of wild-type tryptophan synthase and the alpha-subunit substrate indole-3-glycerol phosphate (IGP) and determined its structure to 1. 8 A resolution. In addition, we determined the 1.4 A resolution structure of the complex with indole-3-propanole phosphate (IPP), a noncleavable IGP analogue. The interaction of the 3'-hydroxyl of IGP with the catalytic alphaGlu49 leads to a twisting of the propane chain and to a repositioning of the indole ring compared to IPP. Concomitantly, the catalytic alphaAsp60 rotates resulting in a translocation of the COMM domain [betaGly102-betaGly189, for definition see Schneider et al. (1998) Biochemistry 37, 5394-5406] in a direction opposite to the one in the IPP complex. This results in loss of the allosteric sodium ion bound at the beta-subunit and an opening of the beta-active site, thereby making the cofactor pyridoxal 5'-phosphate (PLP) accessible to solvent and thus serine binding. These findings form the structural basis for the information transfer from the alpha- to the beta-subunit and may explain the affinity increase of the beta-active site for serine upon IGP binding.     

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.     

Stereochemistry and mechanism of reactions catalyzed by tryptophanase Escherichia coli.

Several beta replacement and alpha,beta elimination reactions catalyzed by tryptophanase from Escherichia coli are shown to proceed stereospecifically with retention of configuration. These conversions include synthesis of tryptophan from (2S,3R)- and (2s,3s)-[3(-3H)]serine in the presence of indole, deamination of these serines in D2O to pyruvate and ammonia, and cleavage of (2S,3R)-and (2S,3S)-[3(-3H)]tryptophan in D2O to indole, pyruvate, and ammonia. A coupled reaction with lactate dehydrogenase was used to trap the stereospecifically labeled [3-H,2H,3H]pryuvates as lactate, which was oxidized to acetate for chirality analysis of the methyl group. During deamination of tryptophan there is significant intramolecular transfer of the alpha proton of the amino acid to C-3 of indole. To determine the exposed face of the cofactor.substrate complex on the enzyme surface and to analyze its conformational orientation, sodium boro[3H]hydride was used to reduce tryptophanase-bound alaninepyridoxal phosphate Schiff's base. Degradation of the resulting pyridoxylalanine to (2S)-[2(-3H)]alanine and (4'S)-[4'(-3H)]pyridoxamine demonstrates that reduction occurs from the exposed si face at C-4' of the complex and that the ketimine double bond is trans.