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.     

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.     

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.     

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

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

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.     

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.     

Affinity chromatography of tryptophan synthase from Escherichia coli. Systematic studies with immobilized tryptophanol phosphate.

Inhibition studies and affinity chromatography indicate that derivatives of tryptophanol phosphate are suitable ligands for the affinity chromatography of tryptophan synthase. A phenyl group on the spacer arm strengthens the interaction of immobilized tryptophanol phosphate with the enzyme. The alpha 2 beta 2 complex specifically requires the presence of 0.3--0.5 M phosphate ions for binding. The alpha subunit binds in dilute Tris buffer, but its binding is also enhanced by the presence of phosphate ions. The beta 2 subunit binds unspecifically but strongly to the affinity material and to a variety of other immobilized hydrophobic ligands. Binding studies with suspensions of affinity material show that the alpha subunit interacts rapidly and reversibly. Indoleglycerol phosphate and indolepropanol phosphate release bound alpha 2 beta 2 complex and alpha subunit in a competitive manner, indicating that the interaction occurs biospecifically, i.e. via the active site of alpha subunit. L-Serine is a non-competitive inhibitor of binding. These results are discussed with regard to the composite-active-site hypothesis [T. E. Creighton (1970) Eur. J. Biochem, 13, 1--10]. Both the alpha subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli can be obtained with high yields and in homogenous form by absorption to the affinity material from partially purified preparations. Elution is achieved with linear gradients either of indolepropanol phosphate or of indoleglycerol phosphate or, in the case of the complex, of L-serine. At the low concentrations of the complex found in crude extracts of wild-type E. coli cells, the unexpectedly high affinity of the beta 2 subunit for hydrophobic ligands leads to partial dissociation of the complex.     

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)     

Microspectrophotometric studies on single crystals of the tryptophan synthase alpha 2 beta 2 complex demonstrate formation of enzyme-substrate intermediates

Microspectrophotometry of single crystals of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium is used to compare the catalytic and regulatory properties of the enzyme in the soluble and crystalline states. Polarized absorption spectra demonstrate that chromophoric intermediates are formed between pyridoxal phosphate at the active site of the beta subunit and added substrates, substrate analogs, and reaction intermediate analogs. Although the crystalline and soluble forms of the enzyme produce some of the same enzyme-substrate intermediates, including Schiff base and quinonoid intermediates, in some cases the equilibrium distribution of these intermediates differs in the two states of the enzyme. Ligands which bind to the active site of the alpha subunit alter the distribution of intermediates formed at the active site of the beta subunit in both the crystalline and soluble states. The three-dimensional structures of the tryptophan synthase alpha 2 beta 2 complex and of a derivative with indole-3-propanol phosphate bound at the active site of the alpha subunit have recently been reported (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 264, 17857-17871). Our present findings help to establish experimental conditions for selecting defined intermediates for future x-ray crystallographic analysis of the alpha 2 beta 2 complex with ligands bound at the active sites of both alpha and beta subunits. These crystallographic studies should explain how catalysis occurs at the active site of the beta subunit and how the binding of a ligand to one active site affects the binding of a ligand to the other active site which is 25 A away.     

The crystal structure of methylglyoxal synthase from Escherichia coli.

BACKGROUND: The reaction mechanism of methylglyoxal synthase (MGS) is believed to be similar to that of triosephosphate isomerase (TIM). Both enzymes utilise dihydroxyacetone phosphate (DHAP) to form an enediol(ate) phosphate intermediate as the first step of their reaction pathways. However, the second catalytic step in the MGS reaction pathway is characterized by the elimination of phosphate and collapse of the enediol(ate) to form methylglyoxal instead of reprotonation to form the isomer glyceraldehyde 3-phosphate. RESULTS: The crystal structure of MGS bound to formate and substoichiometric amounts of phosphate in the space group P6522 has been determined at 1.9 A resolution. This structure shows that the enzyme is a homohexamer composed of interacting five-stranded beta/alpha proteins, rather than the hallmark alpha/beta barrel structure of TIM. The conserved residues His19, Asp71, and His98 in each of the three monomers in the asymmetric unit bind to a formate ion that is present in the crystallization conditions. Differences in the three monomers in the asymmetric unit are localized at the mouth of the active site and can be ascribed to the presence or absence of a bound phosphate ion. CONCLUSIONS: In agreement with site-directed mutagenesis and mechanistic enzymology, the structure suggests that Asp71 acts as the catalytic base. Further, Asp20 and Asp101 are involved in intersubunit salt bridges. These salt bridges may provide a pathway for transmitting allosteric information.     

Structural insights into the mechanism of the PLP synthase holoenzyme from Thermotoga maritima

Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B6 and is an important cofactor for several of the enzymes involved in the metabolism of amine-containing natural products such as amino acids and amino sugars. The PLP synthase holoenzyme consists of two subunits: YaaD catalyzes the condensation of ribulose 5-phosphate, glyceraldehyde-3-phosphate, and ammonia, and YaaE catalyzes the production of ammonia from glutamine. Here we describe the structure of the PLP synthase complex (YaaD-YaaE) from Thermotoga maritima at 2.9 A resolution. This complex consists of a core of 12 YaaD monomers with 12 noninteracting YaaE monomers attached to the core. Compared with the previously published structure of PdxS (a YaaD ortholog in Geobacillus stearothermophilus), the N-terminus (1-18), which includes helix alpha0, the beta2-alpha2 loop (46-56), which includes new helix alpha2a, and the C-terminus (270-280) of YaaD are ordered in the complex but disordered in PdxS. A ribulose 5-phosphate is bound to YaaD via an imine with Lys82. Previous studies have demonstrated a similar imine at Lys149 and not at Lys81 (equivalent to Lys150 and Lys82 in T. maritima) for the Bacillus subtilis enzyme suggesting the possibility that two separate sites on YaaD are involved in PLP formation. A phosphate from the crystallization solution is found bound to YaaD and also serves as a marker for a possible second active site. An ammonia channel that connects the active site of YaaE with the ribulose 5-phosphate binding site was identified. This channel is similar to one found in imidazole glycerol phosphate synthase; however, when the beta-barrels of the two complexes are superimposed, the glutaminase domains are rotated by about 180 degrees with respect to each other.     

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.     

Half-of-the-sites reactivity and all-of-the-sites substrate binding in transaldolase

Transaldolase from Candida utilis is a dimeric protein composed of two identical subunits. The cleavage of fructose 6-phosphate by this enzyme was followed in a rapidmixing spectrophotometer. A very rapid reaction was observed during which 1 mol of glyceraldehyde 3-phosphate/mol of enzyme was released, followed by a much slower reaction in which additional glyceraldehyde 3-phosphate was formed. Binding studies carried out with the same substrate showed that two equivalents of dihydroxyacetone were bound. These results indicate that both sites are active, but that only one functions in the rapid catalytic reaction. The half-of-the-sites reactivity of transaldolase may be attributed to a high degree of negative cooperativity between the two subunits.     

Enzymatic properties of mutant Escherichia coli tryptophan synthase alpha-subunits

Thirty-nine mutant tryptophan synthase alpha subunits have been purified and analyzed (in the presence of the beta 2-subunit) for their enzymatic (kcat, Km) behavior in the reactions catalyzed by the alpha 2.beta 2 complex, the fully constituted form of this enzyme. The mutant alpha subunits, obtained by in vitro random, saturation mutagenesis of the encoding trpA gene, contain single amino acid substitutions at sites within the first 121 residues of the alpha polypeptide. Four categories of altered residues have been tentatively assigned roles in the catalytic functions of this enzyme: 1) catalytic residues (Glu49 and Asp60); 2) residues involved in substrate binding or orientation (Phe22, Thr63, Gln65, Tyr102, and Leu105); 3) residues involved in alpha.beta subunit interactions (Gly51, Pro53, Asp56, Asp60, Pro62, Ala67, Phe72, Thr77, Pro78, Tyr102, Asn104, Leu105, and Asn108); and 4) residues with no apparent catalytic roles. Catalytic residue alterations result in no detectable activity in the alpha-subunit specific reactions. Substrate binding/orientation roles are detected enzymatically primarily as rate defects; alterations only at Tyr102 result in apparent Km effects. alpha.beta interaction roles are detected as rate defects in all tryptophan synthase reactions plus Km increases for the alpha-subunit substrate, indole-3-glycerol phosphate, only when L-serine is present at the beta 2-subunit active site. A substitution at only one site, Asn104, appears to be unique in its potential effect on intersubunit channeling of indole, the product of the alpha-subunit specific reaction, to the beta 2-subunit active site.     

Photoinactivation and photoaffinity labeling of tryptophan synthase alpha 2 beta 2 complex by the product analogue 6-azido-L-tryptophan

The photoaffinity reagent 6-azido-L-tryptophan was synthesized by chemical methods. It binds reversibly in the dark to the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli and forms a quinonoid intermediate with enzyme-bound pyridoxal phosphate (lambda max = 476 nm). The absorbance of this chromophore has been used for spectrophotometric titrations to determine the binding of 6-azido-L-tryptophan (the half-saturation value [S]0.5 = 6.3 microM). Photolysis of the quinonoid form of the alpha 2 beta 2 complex results in time-dependent inactivation of the beta 2 subunit but not of the alpha subunit. The extent of photoinactivation is directly proportional to the absorbance at 476 nm of the quinonoid intermediate prior to photolysis. The substrate L-serine is a competitive inhibitor of 6-azido-L-tryptophan binding and photoinactivation. The competitive inhibitors L-tryptophan, D-tryptophan, and oxindolyl-L-alanine also protect against photoinactivation. The results demonstrate that 6-azido-L-tryptophan is a quasi-substrate for the alpha 2 beta 2 complex of tryptophan synthase and that photolysis of the enzyme-quasi-substrate quinonoid intermediate results in photoinactivation. The modified alpha 2 beta 2 complex retains its ability to bind pyridoxal phosphate and to cleave indole-3-glycerol phosphate, a reaction catalyzed by the alpha subunit. 6-Azido-L-tryptophan (side-chain 1,2,3-14C3 labeled) was synthesized enzymatically from 6-azidoindole and uniformly labeled L-[14C]serine by the alpha 2 beta 2 complex of tryptophan synthase on a preparative scale and has been isolated. Incorporation of 14C label from 6-azido-L-[14C]tryptophan is stoichiometric with inactivation. Our finding that most of the incorporated 14C label is bound in an unstable linkage suggests that an active site carboxyl residue is the major site of photoaffinity labeling by 6-azido-L-tryptophan.     

Mechanism of mutual activation of the tryptophan synthase alpha and beta subunits. Analysis of the reaction specificity and substrate-induced inactivation of active site and tunnel mutants of the beta subunit

The origin of reaction and substrate specificity and the control of activity by protein-protein interaction are investigated using the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. We have compared some spectroscopic and kinetic properties of the wild type beta subunit and five mutant forms of the beta subunit that have altered catalytic properties. These mutant enzymes, which were engineered by site-directed mutagenesis, have single amino acid replacements in either the active site or in the wall of a tunnel that extends from the active site of the alpha subunit to the active site of the beta subunit in the alpha 2 beta 2 complex. We find that the mutant alpha 2 beta 2 complexes have altered reaction and substrate specificity in beta-elimination and beta-replacement reactions with L-serine and with beta-chloro-L-alanine. Moreover, the mutant enzymes, unlike the wild type alpha 2 beta 2 complex, undergo irreversible substrate-induced inactivation. The mechanism of inactivation appears to be analogous to that first demonstrated by Metzler's group for inhibition of two other pyridoxal phosphate enzymes. Alkaline treatment of the inactivated enzyme yields apoenzyme and a previously described pyridoxal phosphate derivative. We demonstrate for the first time that enzymatic activity can be recovered by addition of pyridoxal phosphate following alkaline treatment. We conclude that the wild type and mutant alpha 2 beta 2 complexes differ in the way they process the amino acrylate intermediate. We suggest that the wild type beta subunit undergoes a conformational change upon association with the alpha subunit that alters the reaction specificity and that the mutant beta subunits do not undergo the same conformational change upon subunit association.     

Tryptophan synthase, an allosteric molecular factory

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

A new arrangement of (beta/alpha)8 barrels in the synthase subunit of PLP synthase

Pyridoxal 5'-phosphate (PLP, vitamin B6), a cofactor in many enzymatic reactions, has two distinct biosynthetic routes, which do not coexist in any organism. Two proteins, known as PdxS and PdxT, together form a PLP synthase in plants, fungi, archaea, and some eubacteria. PLP synthase is a heteromeric glutamine amidotransferase in which PdxT produces ammonia from glutamine and PdxS combines ammonia with five- and three-carbon phosphosugars to form PLP. In the 2.2-A crystal structure, PdxS is a cylindrical dodecamer of subunits having the classic (beta/alpha)8 barrel fold. PdxS subunits form two hexameric rings with the active sites positioned on the inside. The hexamer and dodecamer forms coexist in solution. A novel phosphate-binding site is suggested by bound sulfate. The sulfate and another bound molecule, methyl pentanediol, were used to model the substrate ribulose 5-phosphate, and to propose catalytic roles for residues in the active site. The distribution of conserved surfaces in the PdxS dodecamer was used to predict a docking site for the glutaminase partner, PdxT.     

The tryptophan synthase alpha 2 beta 2 complex. Cleavage of a flexible loop in the alpha subunit alters allosteric properties

This study explores the catalytic and allosteric roles of a flexible loop in tryptophan synthase. Trypsin is known to cleave the tryptophan synthase alpha 2 beta 2 complex in an alpha subunit loop at Arg-188. Cleavage yields an active "nicked" alpha 2 beta 2 derivative. The new results provide evidence that the alpha subunit loop serves two important roles: substrate binding and communicating the effects of substrate binding to the beta subunit. A role for the loop in substrate binding is supported by our finding that addition of a substrate analogue of the alpha subunit, alpha-glycerol 3-phosphate, decreases the rate of cleavage by trypsin. An allosteric role for the loop is supported by the finding although the native alpha 2 beta 2 complex is strongly inhibited by alpha-glycerol 3-phosphate, the nicked alpha 2 beta 2 complex is desensitized to this inhibition. The time course of proteolysis in the presence and absence of alpha-glycerol 3-phosphate is followed by sodium dodecyl sulfate-gel electrophoresis and by assays of activity in the presence and absence of alpha-glycerol 3-phosphate. We use spectroscopic measurements of the pyridoxal phosphate-L-tryptophan intermediates at the active site of the beta subunit to determine the affinity of the native and nicked enzymes for L-tryptophan and alpha-glycerol 3-phosphate. Although cleavage alters the equilibrium distribution of intermediates and reduces the affinity for alpha-glycerol 3-phosphate, it has little effect on the affinity for amino acids bound to the beta subunit. We conclude that the loop in the alpha subunit is important for ligand binding and for communicating the effects of ligand binding from the alpha subunit to the beta subunit in the alpha 2 beta 2 complex.     

Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium

The three-dimensional structure of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium has been determined by x-ray crystallography at 2.5 A resolution. The four polypeptide chains are arranged nearly linearly in an alpha beta beta alpha order forming a complex 150 A long. The overall polypeptide fold of the smaller alpha subunit, which cleaves indole glycerol phosphate, is that of an 8-fold alpha/beta barrel. The alpha subunit active site has been located by difference Fourier analysis of the binding of indole propanol phosphate, a competitive inhibitor of the alpha subunit and a close structural analog of the natural substrate. The larger pyridoxal phosphate-dependent beta subunit contains two domains of nearly equal size, folded into similar helix/sheet/helix structures. The binding site for the coenzyme pyridoxal phosphate lies deep within the interface between the two beta subunit domains. The active sites of neighboring alpha and beta subunits are separated by a distance of about 25 A. A tunnel with a diameter matching that of the intermediate substrate indole connects these active sites. The tunnel is believed to facilitate the diffusion of indole from its point of production in the alpha subunit active site to the site of tryptophan synthesis in the beta active site and thereby prevent its escape to the solvent during catalysis.