Subunit Of Tryptophan Synthase Research Articles

The Quarternary Structure of Tryptophan Synthase from Escherichia coli

Single cysteine residues on the separated α and β2 subunits of tryptophan synthase were labelled with various fluorescent ligands suitable for fluorescence energy transfer measurements. Labelled α subunits, reconstituted into an αL2β2 complex, retained more than 75% activity. Labelled β2 subunits, either free or reconstituted into an α2βL2′ complex, retained 10–30% of their activity, depending on the label. The coenzyme pyridoxal 5′‐phosphate served as a natural fluorescent label of the active site of the β2 subunit. Different combinations of labelled subunits resulted in doubly labelled αL2βL2′ complexes that allowed five independent distances to be estimated by fluorescence energy transfer: within one β protomer, CysL–pyridoxal 5′‐phosphate = 1.4–3.2 nm; between two β protomers, CysL–CysL= 0.5–2.3 nm; between one α protomer and a β protomer, CysL–pyridoxal 5′‐phosphate = 1.9–3.2 nm, CysL–CysL= 3.1–4.4 nm; between two α protomers, CysL–CysL= 3.1–3.9 nm. Translational frictional ratios obtained from measurements of sedimentation and diffusion constants and partial specific volumes provided independent information on the shapes of the different particles in solution. Both the α and the β protomer are known to consist of two globular domains of known molecular weight. These were assumed to be approximately spherical and used to construct models first of the α and β2 subunits and then of the α2β2 complex. The data on specific distances and shape factors, together with considerations of symmetry and compactness allow many possible spatial arrangements of the domains to be eliminated. The radii of gyration, maximum dimensions and hydrated volumes calculated for the best models of the α and β2 subunits and the α2β2 complex agree reasonably well with the results of independent small‐angle X‐ray scattering studies [Wilhelm et al. (1982) Eur. J. Biochem. 129, 51–56. The active sites responsible for cleavage of indoleglycerol phosphate to indole and for condensation of indole with L‐serine are approximately 2 nm apart, ruling out the composite active sites hypothesis.

Small-angle X-ray scattering studies of tryptophan synthase from Escherichia coli and its alpha and beta 2 subunits.

The alpha and beta 2 subunits of tryptophan synthase were investigated by small-angle X-ray scattering. The molecular parameters are: radius of gyration, alpha: 1.95 nm, beta 2: 3.01 nm; maximum particle diameter, alpha: 5.8 nm, beta 2: 10.5 nm; and hydrated volume, alpha: 60 nm3, beta 2 160 nm3. The shape of the alpha subunit can best be described by a circular cylinder, slightly tapered at one end. An elongated elliptical cylinder with its cross section larger in the middle than at the ends was found to be a model equivalent in scattering to the beta 2 subunits. The alpha 2 beta 2 enzyme complex was found to have a radius of gyration of 4.01 nm, a maximum length of 13.5 nm, and a hydrated volume of 270 nm3. No satisfactory fit of the scattering data was obtainable by mere apposition of the models of the alpha and beta 2 subunits. Two cylinders overlapping laterally fit the experimental data considerably better, suggesting changes in the conformation of the subunits on forming the alpha 2 beta 2 complex.

Guanidine hydrochloride induced unfolding of the alpha subunit of tryptophan synthase and of the two alpha proteolytic fragments: evidence for stepwise unfolding of the two alpha domains.

The relationship between the domain structure of the alpha subunit of Escherichia coli tryptophan synthase and the mechanism of unfolding of the alpha subunit is investigated. Previous studies of the unfolding of the alpha subunit by increasing concentrations of guanidine hydrochloride or urea detected a partially unfolded form of the alpha subunit at intermediate concentrations of either denaturant. The possibility that this partially unfolded form of the alpha subunit results from the preferential unfolding of one of the two domains of the alpha subunit is now investigated. This study utilizes two proteolytic fragments of the alpha subunit, alpha-1 and alpha-2, which have been shown to refold independently and to correspond to two domains of the alpha subunit. The effects of guanidine hydrochloride concentration on the separate alpha-1 and alpha-2 fragments, on the intact alpha subunit, and on the derivative nicked by trypsin (alpha') are compared by measuring ellipticity at 222 nm and by measuring the susceptibility of tyrosyl residues to chemical modification. The results show that guanidine hydrochloride induced unfolding of the alpha subunit results from the stepwise unfolding of the two domains: the alpha-2 fragment and the corresponding domain in the intact alpha subunit are unfolded by low concentrations of guanidine hydrochloride; the alpha-1 fragment and the corresponding domain in the intact alpha subunit are unfolded by higher concentrations of guanidine hydrochloride.

Circular dichroism studies on the interaction of tryptophan synthase with pyridoxal 5'-phosphate.

The interaction between the beta 2 subunit of tryptophan synthase and the coenzyme pyridoxal 5'-phosphate (PLP) is characterized by induced circular dichroism (CD) in the near-UV (260-285 nm) and in the visible region (320-480 nm, extrinsic Cotton effect). Because of its high mean residue ellipticity ([theta] = 56 deg cm2 dmol-1 for the isolated holo-beta 2 subunit and 102 deg cm2 dmol-1 for the alpha 2-holo-beta 2 complex, respectively) the latter has been used to define different conformational states of the beta 2 dimer via CD titrations. Fitting the obtained binding parameters to the known data from equilibrium dialysis leads to the result that the low-affinity state of the isolated beta 2 subunit shows a 3 times greater rotational strength than the holoenzyme in the high-affinity state. The generation of the final CD amplitude is characterized by a rate constant intermediate between the values for the formation of the internal aldimine and for the regain of enzymatic activity. Interaction of the alpha 2-apo-beta 2 bienzyme complex with the cofactor leads to a hyperbolic binding curve which is apparently free of contributions caused by unspecific PLP binding outside the active center. The determined dissociation constant of 9 x 10(-7) M is in good agreement with the value of 1 x 10(-6) M as obtained by equilibrium dialysis. Binding kinetics reveal a very slow process with a rate constant of 2.6 x 10(-4) s-1, significantly smaller than that for the regain of catalytic activity during reconstitution of the enzyme.

Characterization of the slow steps in the folding of the alpha subunit of tryptophan synthase.

Characterization of the slow steps in the folding of the alpha subunit of tryptophan synthase.

Urea-induced unfolding of the alpha subunit of tryptophan synthase: evidence for a multistate process.

The urea-induced unfolding of the alpha subunit of tryptophan synthase from E. coli was monitored by optical spectroscopy and by urea-gradient gel electrophoresis. Three independent lines of evidence support the conclusion that one or more stable intermediates are present in this process: (i) Satisfactory fits of the equilibrium unfolding transitions obtained from difference spectroscopy at 286 nm and circular dichroism spectroscopy at 222 nm require a model which involves a stable intermediate in addition to the native and unfolded forms. (ii) Kinetic studies of the change in the extinction coefficient at 286 nm show that while the unfolding is well described by a single exponential change the refolding kinetics are complex. The nature of the dependence of the refolding kinetics on the initial concentration of urea supports the conclusion that at least one stable intermediate exists. (iii) The patterns obtained from urea-gradient gel electrophoresis experiments on the alpha subunit show that at least one and possibly two stable intermediates are involved; the intermediates have markedly different degrees of compactness. A kinetic model for the folding of the alpha subunit, consistent with all of these results, can be formulated.

Kinetics of cooperative ligand binding to the apo beta 2 subunit of tryptophan synthase and its modulation by the alp ha subunit.

The different binding mechanisms of pyridoxine 5'-phosphate and N-phophopridoxyl-L-serine have been investigated by kinetic studies with rapid reaction techniques. Pyridoxine 5'-phosphate binds in a single rapid step to the alpha 2 apo beta 2 complex and in a single slow step to the nicked apo beta 2 subunit that is obtained by limited proteolysis with trypsin. Both pyridoxine 5'-phosphate and N-phosphopyridoxyl-L-serine bind to the apo beta 2 subunit with a comparatively slow binding step, followed by an event slower isomerization reaction. These findings are consistent with nonexclusive concerted mechanism of cooperative binding but cannot be explained by the simple sequential mechanism. A quantitative fit of the rate and equilibrium data to the concerted mechanism generally yielded the pertinent rate and equilibrium constants. In particular, the same value of L0 = [T0]/[R0] = 200 +/- 50 simultaneously satisfies the data obtained with three different ligands. The comparison of the mechanisms of ligand binding to the three states of the apo beta 2 subunit suggests that the alpha 2 apo beta 2 complex is similar to the high-affinity R state and the nicked apo beta 2 subunit is similar to the low-affinity T state of the apo beta 2 subunit. The slow isonerization involved in the cooperative binding of the ligands to the intact apo beta 2 subunit is discussed in terms of local and concerted conformational changes involving the two autonomously folding domains of the beta protomer.

Mechanism of reconstitution of the apo beta 2 subunit and the alpha 2 apo beta 2 complex of tryptophan synthase with pyridoxal 5'-Phosphate: kinetic studies.

The mechanism of pryidoxal 5'-phosphate (PLP) binding to both the alpha apo beta 2 complex and the apo beta 2 subunit of tryptophan synthase was investigated by rapid mixing experiments. Absorption and fluorescence changes were used to monitor the binding reaction directly. Reduction with sodium borohydride provided the rate of formation of the internal aldimine with the lysine amino group of the enzyme, and substrate turnover monitored the rate of formation of active enzyme. The alpha 2 apo beta 2 complex binds PLP in a sequence of three steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by formation of an active alpha 2 holo beta 2 complex. The two binding sites appear to bind PLP independently. The apo beta 2 subunit binds PLP cooperatively in a sequence of three steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by the formation of the enzymically active holo beta 2 subunit. Taken together with kinetic studies of pyridoxine phosphate binding [Tschopp, J., & Kirschner, K. (1980) Biochemistry (second paper of three in this issue)], the rate data of the apo beta 2 subunit are shown to be consistent with the concerted mechanism. The difference between the values of the isomerization rate constants of bound PLP and bound PNP appear to result from the covalent internal aldimine, which is formed with PLP but not with PNP.

Location of the reactive sulfhydryl residues in the primary sequence of the β 2 subunit of tryptophan synthase of [formula omitted

Two of the 5 sulfhydryl residues of the β 2 subunit of tryptophan synthase have previously been shown to react with N-ethylmaleimide and to have active site roles. We now show that the single sulfhydryl which reacts with N-ethylmaleimide in the presence of pyridoxal phosphate is cysteine-170. The essential sulfhydryl which reacts with N-ethylmaleimide or with 2-nitro-5-thiocyanobenzoic acid after removal of pyridoxal phosphate is cysteine-230. The affinity reagent, bromoacetylpyridoxamine phosphate, reacts variably with cysteine-62 or with cysteine-230.

Effect of single amino acid substitutions on the thermal stability of the alpha subunit of tryptophan synthase.

Effect of single amino acid substitutions on the thermal stability of the alpha subunit of tryptophan synthase.

An active proteolytic derivative of the .alpha. subunit of tryptophan synthase. Identification of the site of cleavage and characterization of the fragments

An active proteolytic derivative of the .alpha. subunit of tryptophan synthase. Identification of the site of cleavage and characterization of the fragments

Subunit interaction in tryptophan synthase of Escherichia coli: calorimetric studies on association of .alpha. and .beta.2 subunits

Association of the apo-beta 2 and the holo-(beta-PLP)2 subunits of tryptophan synthase from Escherichia coli (L-serine hydro-lyase (adding indole) (EC 4.2.1.20)) with alpha subunits of the same enzyme has been studied by microcalorimetry. The results obtained from thermometric titrations clearly demonstrate that only the native complex alpha2beta 2 is formed, independent of an excess of alpha protein. The reaction of the holo-(beta-PLP)2 with alpha subunits at 25 degrees C is accompanied by a negative enthalpy change, which is almost twice as large as that for complex formation with the apo-beta 2 protein, thus indicating that the interaction enthalpy becomes more favorable in the presence of the coenzyme pyridoxal 5'-phosphate (PLP). Both reaction enthalpies show very large negative temperature coefficients, -3600 +/- 100 cal K-1 (Mol of beta 2)-1 being the value for the formation of the apoenzyme and -2300 +/- 100 cal K-1 (mol of beta 2)-1 pertaining to formation of the holoenzyme. The studies on the association of alpha and beta2 subunits in the two buffers revealed that at 25 degrees C approximately 0.75 proton are absorbed in the presence and absence of the coenzyme, whereas at 35 degrees C one proton is taken up from the solution when PLP is present, but two if the apo-beta 2 complex reacts. These results are a clear indication of energetic linkage between intersubunit interaction, hydrogen ion equilibria, and the binding of the coenzyme.

Cooperative Binding of α Subunits to the Apo‐β2 Subunit of Tryptophan Synthase from Escherichia coli

The binding parameters for the interaction of α subunits with the dimeric apo‐β2 subunit of tryptophan synthase from Escherichia coli were determined by gel chromatography, equilibrium dialysis and sedimentation velocity experiments. In 0.1 M pyrophosphate buffer pH 7.5 the binding of two α subunits to the stable apo‐β2 dimer is cooperative. Fitting the data to the Adair equation yielded the apparent microscopic dissociation constants for the complexes with one and two bound α chains. Since they differ significantly, the apo‐β2 dimer seems to exist in distinct conformational states depending on the saturation with α subunits.This finding is in agreement with a recently published scheme summarizing the interactions between the apo‐β2 subunit and its ligands, α subunit and pyridoxal 5′‐phosphate. The binding between the subunits shows a moderate temperature‐dependence, suggesting that hydrophobic effects may be involved in the subunit association to form the apo‐multienzyme complex.

Affinity labeling of the pyridoxal phosphate binding site of the beta2 subunit of Escherichia coli tryptophan synthase

We have synthesized bromoacetylpyridoxamine phosphate and bromoacetylpyridoxamine and have shown that they meet three criteria for affinity labels of the beta2 subunit of tryptophan synthase: (i) the kinetic data of inactivation indicate that a binary complex is formed prior to covalent attachment; (ii) inactivation is largely prevented by the presence of pyridoxal phosphate; and (iii) inactivation is stoichiometric with incorporation of 0.7 to 0.8 mol of chromophore/mol of beta monomer. Our conclusion that inactivation of the apo beta2 subunit by bromoacetylpyridoxamine phosphate is due to the modification of cysteine is based on the disappearance of 1 mol of -SH/beta monomer and on the finding that [14C]carboxymethyl derivative in the acid hydrolysate of the protein modified by bromo[14C]acetylpyridixamine phosphate. A 39-residue tryptic peptide containing this essential cysteine has been isolated and purified from the bromo[14C]acetylpyridoxamine phosphate-labeled beta2 subunit.

Studies of the function and location of two cysteines in the β 2 subunit of tryptophan synthase

Our findings that the apo β 2 subunit of tryptophan synthase of Escherichia coli is inactivated by the modification of one sulfhydryl residue per monomer by nitrothiocyanobenzoic acid and is reactivated by removal of the CN group indicate that the reactive sulfhydryl residue (SH-I) is essential for catalytic activity. SH-I is shown to be the same residue which was previously found to react with bromoacetylpyridoxamine phosphate and different from a sulfhydryl (SH-II) which reacts with N-ethylmaleimide in the presence of pyridoxal phosphate. The results of partial tryptic digestions of β 2 subunit labeled selectively at SH-I or SH-II show that both sulfhydryl residues are located in the F 1 fragment which also contains the pyridoxal phosphate binding site.

Regulation of enzyme synthesis in the tryptophan pathway of Acinetobacter calcoaceticus.

In Acinetobacter calcoaceticus the seven genes coding for the enzymes responsible for tryptophan synthesis map at three chromosomal locations. Two three-gene clusters, one (trpGDC) specifying the small subunit of anthranilate synthase, phosphoribosyl transferase, and indoleglycerol phosphate synthase and the other (trpFBA) specifying phosphoribosyl anthranilate isomerase and both tryptophan synthase subunits, are not linked to each other or to the trpE gene specifying the large anthranilate synthase subunit. When regulation of trp gene expression is studied in the wild type, only the level of the trpF gene product decreases upon addition of tryptophan to the medium. Tryptophan starvation of tryptophan auxotrophs, however, results in increased levels of all the tryptophan enzymes; this and additional evidence suggests that the expression of all the trp genes is subject to repression. The trpGDC genes are coordinately controlled, and the trpE gene is regulated in parallel with them. The trpFBA genes are controlled neither coordinately nor in parallel with the other trp genes, but respond proportionally when compared with each other. So far, two types of constitutive mutants have been found. The first class of mutants apparently occurs in the structural gene for a repressor protein; this repressor locus is unlinked to any of the biosynthetic trp genes and affects only the expression of trpE and the trpGDC cluster. The second class contains mutants closely linked to the trpGDC region; they overproduce only the gene products of this cluster.

Steady‐State Kinetic Studies of the Synthesis of Indoleglycerol Phosphate Catalyzed by the α Subunit of Tryptophan Synthase from Escherichia coli

For the alpha subunit of tryptophan synthase and at constant concentration of D-glyceraldehyde 3-phosphate the saturation curves with respect to indole concentration are weakly sigmoidal. This phenomenon can be explained by interaction between indole bound to the effector site established previously and the active center of the monomeric alpha subunit. Kinetic studies of the inhibition of indoleglycerol phosphate synthesis by the analogue indolepropanol phosphate show that the inhibition is competitive with respect to D-glyceraldehyde 3-phosphate and non-competitive with respect to indole. Mechanisms with random addition of substrates or ordered addition with indole binding first can therefore be excluded. A quantitative fit of the data has been obtained to an ordered addition mechanism with D-glyceraldehyde 3-phosphate binding first and with a distribution of the enzyme between two states differing in V, governed by the binding of indole to the effector site. The kinetic constants obtained for the alpha subunit have been compared with those of the alpha 2 beta 2 complex of tryptophan synthase. Protein-protein interaction of the alpha subunit with the beta 2 subunit (a) does not alter the catalytic of the indoleglycerol phosphate synthesis, (b) suppresses the substrate activation by indole, and (c) changes the various equilibrium, rate and steady-state constants in the sense of conveying higher substrate specificity and catalytic efficiency to the alpha-subunit. The occurrence of local and gross conformational changes in the tryptophan synthase system is discussed.

Circular dichroism and fluorescence studies on the binding of ligands to the alpha subunit of tryptophan synthase.

Binding to the alpha subunit of tryptophan synthase induces extrinsic Cotton effects in the substrates indole (IND), indoleglycerol phosphate (IGP), and D-glyceraldehyde-3-P (D-GAP) and in the inhibitor indolepropanol phosphate (IPP). These effects disappear when the enzyme is denatured in guanidinium chloride. The induced circular dichroism (CD) was used to determine the dissociation constant and the number of binding sites for IPP. The dissociation constant so determined is equal to 48 muM and is in good agreement with the value of 48 muM obtained by equilibrium dialysis. From the temperature dependence of the dissociation constant, a value of -2.8 kcal/mol for the binding enthalpy was obtained. The determination of dissociation constants by means of extrinsic Cotton effects is shown to be quite feasible. CD competition experiments with glycerol phosphate (GP) suggest that IPP binds bifunctionally to the enzyme: via its indole part and its phosphate group. Indolepropanol, which lacks the phosphate group, does not show an extrinsic Cotton effect. Since the induced CD is strongly dependent on the binding geometry, the close similarity between the induced spectra in IPP and IGP is additional evidence that IPP is a good substrate analog. Binding to the enzyme results in a blue shift of the IPP fluorescence emission maximum. The dissociation constant determined by fluorescence titration equals 46 muM and agrees well with the values determined by the other two methods. Previous biochemical and fast kinetic studies suggested the existence of multiple conformational states for the enzyme and of ligand-induced conformational changes. No evidence was found in the far-uv CD spectra for conformational changes upon binding of IND and D-GAP. For IPP a very small effect was observed.

Immunochemical and enzymatic comparisons of the tryptophan synthase alpha subunits from five species of Enterobacteriaceae.

The reactive surface structures of alpha subunits of tryptophan synthase from Escherichia coli, Shigella dysenteriae, Salmonella typhimurium, Aerobacter aerogenes, and Serratia marcescens were compared by measuring (i) their reactivities in micro-complement-fixation assays with antibodies directed specifically to E. coli wild-type alpha subunit, (ii) their reactivities in enzyme neutralization assays with the same antibodies, and (iii) their binding affinities for tryptophan synthase beta(2) subunits. The enzymes from the four heterologous species cross-reacted in the microcomplement-fixation assays with the anti-E. coli alpha subunit antibodies, each to a different degree. However, neutralization titers of the antibodies reacting with the various alpha subunits were comparatively similar, and the beta(2) subunit-binding and -stimulating abilities of the alpha subunits were even more closely alike. The results suggested that the tertiary structure of the beta(2) subunit-binding site of the alpha subunit has been conserved, relative to the rest of the molecule, during the evolutionary divergence of the species of Enterobacteriaceae.