Conformational effects of ligand binding on the beta 2 subunit of Escherichia coli tryptophan synthase analyzed with monoclonal antibodies

Five monoclonal antibodies recognizing five different epitopes of the native beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.1.2.20) were used to analyze the conformational changes occurring upon ligand binding or chemical modifications of the enzyme. For this purpose, the affinities of each antibody for the different forms of the enzyme were determined by using an enzyme-linked immunosorbent assay which allows measurement of the dissociation constant of antigen-antibody equilibrium in solution. The fixation of the coenzyme pyridoxal 5'-phosphate and the substrate L-serine modifies the affinity constants of most of the antibodies for the enzyme, thus showing the existence of extended conformational rearrangements of the protein. The association of the alpha subunit with the beta 2 subunit, which brings about an increase of the tryptophan synthase activity and abolishes the serine deaminase activity of beta 2, is accompanied by an important conformational change of the N-terminal domain of beta 2 (F1) since none of the anti-F1 monoclonal antibodies can bind to alpha 2 beta 2. Similarly, chemical modifications of beta 2 which are known to produce significant effects on the enzymatic activities of beta 2 result in changes of the affinities of the monoclonal antibodies which can be interpreted as the acquisition of different conformational states of the enzyme.     

Monoclonal antibodies to rabbit skeletal muscle phosphorylase kinase. Probes for studies of subunit function

Monoclonal antibodies to rabbit skeletal muscle phosphorylase kinase were produced by the conventional hybridoma cell technique. 90 out of 600 hybridomas were found to produce phosphorylase kinase binding antibodies from which only five secreted also phosphorylase kinase activity affecting antibodies. Three of them were cloned; two hybridomas resisted all cloning efforts. Employing immunoblot technique all monoclonal antibodies show cross-reactivity with the alpha, beta, and gamma subunits of phosphorylase kinase indicating that similar, if not identical, epitopes are present on these three subunits. No cross-reactivity with delta is observed. Monoclonal antibodies secreted by two clones which bind to the alpha subunit stimulate the Ca2+-independent A0 activity of phosphorylase kinase more than 30-fold, whereas all other monoclonal antibodies obtained are ineffective in this respect. Monoclonal antibodies binding to the beta subunit inhibit the Ca2+-dependent activities significantly. Antibody produced by one hybridoma binds to the alpha, beta, and gamma subunits with approximately the same affinity. Based on the dual function of calmodulin in phosphorylase kinase (Hessová, Z., Varsányi, M., and Heilmeyer, L.M.G., Jr. (1985) Eur. J. Biochem. 146, 107-115) we conclude that binding of anti-alpha monoclonal antibodies to a regulatory domain in the alpha subunit results in an uncoupling of the inhibitory function of the Ca2+-free delta from the holoenzyme which leads to a concomitant increase in A0 activity. Furthermore, binding of anti-beta monoclonal antibodies to the beta subunit prevents a signal transfer from the Ca2+-saturated delta to the catalytic site of the holoenzyme which inhibits the Ca2+-dependent activities.     

Tryptophan synthase alpha subunit glutamic acid 49 is essential for activity. Studies with 19 mutants at position 49

We have obtained a complete set of 20 variants of the alpha subunit of tryptophan synthase of Escherichia coli at position 49 in order to extend our previous studies on the effects of single amino acid replacements at position 49 on structure and function. Thirteen mutant alpha subunits have been newly constructed by site-directed mutagenesis using oligonucleotides. Six mutants were available from previous studies. We find that the wild type and all of the mutant alpha subunits form alpha 2 beta 2 complexes with the beta 2 subunit of tryptophan synthase with similar association constants and similarly stimulate the activity of the beta 2 subunit in the synthesis of L-tryptophan from L-serine and indole. Thus none of the changes at position 49 produces a change in the conformation of the alpha subunit which significantly interferes with normal subunit interaction. However, the 19 mutant alpha 2 beta 2 complexes are completely devoid of activity in reactions normally catalyzed by the active site of the alpha subunit. This is the first time that these several activities have been measured with a series of highly purified alpha subunits altered by mutation at a single site. Our finding that the mutant in which glutamic acid 49 is substituted by aspartic acid is totally devoid of alpha activity is especially significant and is strong evidence that glutamic acid 49 is an essential catalytic base in the reaction catalyzed by the alpha subunit. This result is consistent with the results of previous genetic studies, with evolutionary comparisons using sequence analysis, and with recent results from x-ray crystallography of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium.     

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.     

Conformational changes induced by domain assembly within the beta 2 subunit of Escherichia coli tryptophan synthase analysed with monoclonal antibodies

The effects of domain assembly on the conformation of the F1 (N-terminal) and F2 (C-terminal) domains of the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were analysed using six monoclonal antibodies which recognize six different epitopes of the native beta 2 subunit (five carried by the F1 domain and one carried by the F2 domain). For this purpose, the affinity constant of each monoclonal antibody for the isolated domains F1 or F2, the associated domains in the trypsin-nicked apo-beta 2 and in the native apo-beta 2 subunits were determined, both with the intact immunoglobulin and the Fab fragment. It was found that the association of the F1 and F2 domains within beta 2 is accompanied by structural changes of the two domains, as detected by variations of their affinity constants for the monoclonal antibodies.     

Conformational Changes in the tryptophan synthase from a hyperthermophile upon alpha2beta2 complex formation: crystal structure of the complex

The three-dimensional structure of the bifunctional tryptophan synthase alpha(2)beta(2) complex from Pyrococcus furiosus was determined by crystallographic analysis. This crystal structure, with the structures of an alpha subunit monomer and a beta(2) subunit dimer that have already been reported, is the first structural set in which changes in structure that occur upon the association of the individual tryptophan synthase subunits were observed. To elucidate the structural basis of the stimulation of the enzymatic activity of each of the alpha and beta(2) subunits upon alpha(2)beta(2) complex formation, the conformational changes due to complex formation were analyzed in detail compared with the structures of the alpha monomer and beta(2) subunit dimer. The major conformational changes due to complex formation occurred in the region correlated with the catalytic function of the enzyme as follows. (1) Structural changes in the beta subunit were greater than those in the alpha subunit. (2) Large movements of A46 and L165 in the alpha subunit due to complex formation caused a more open conformation favoring the entry of the substrate at the alpha active site. (3) The major changes in the beta subunit were the broadening of a long tunnel through which the alpha subunit product (indole) is transferred to the beta active site and the opening of an entrance at the beta active site. (4) The changes in the conformations of both the alpha and beta subunits due to complex formation contributed to the stabilization of the subunit association, which is critical for the stimulation of the enzymatic activities.     

Immunochemical and Enzymatic Comparisons of the Tryptophan Synthase α 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.     

Monoclonal Antibodies to the [alpha]- and [beta]-Subunits of the Plant Mitochondrial F1-ATPase

We have generated nine monoclonal antibodies against subunits of the maize (Zea mays L.) mitochondrial F1-ATPase. These monoclonal antibodies were generated by immunizing mice against maize mitochondrial fractions and randomly collecting useful hybridomas. To prove that these monoclonal antibodies were directed against ATPase subunits, we tested their cross-reactivity with purified F1-ATPase from pea cotyledon mitochondria. One of the antibodies ([alpha]-ATPaseD) cross-reacted with the pea F1-ATPase [alpha]-subunit and two ([beta]-ATPaseD and [beta]-ATPaseE) cross-reacted with the pea F1-ATPase [beta]-subunit. This established that, of the nine antibodies, four react with the maize [alpha]-ATPase subunit and the other five react with the maize [beta]-ATPase subunit. Most of the monoclonal antibodies cross-react with the F1-ATPase from a wide range of plant species. Each of the four monoclonal antibodies raised against the [alpha]-subunit recognizes a different epitope. Of the five [beta]-subunit antibodies, at least three different epitopes are recognized. Direct incubation of the monoclonal antibodies with the F1-ATPase failed to inhibit the ATPase activity. The monoclonal antibodies [alpha]-ATPaseD and [beta]-ATPaseD were bound to epoxide-glass QuantAffinity beads and incubated with a purified preparation of pea F1-ATPase. The ATPase activity was not inhibited when the antibodies bound the ATPase. The antibodies were used to help map the pea F1-ATPase subunits on a two-dimensional map of whole pea cotyledon mitochondrial protein. In addition, the antibodies have revealed antigenic similarities between various isoforms observed for the [alpha]- and [beta]-subunits of the purified F1-ATPase. The specificity of these monoclonal antibodies, along with their cross-species recognition and their ability to bind the F1-ATPase without inhibiting enzymic function, makes these antibodies useful and invaluable tools for the further purification and characterization of plant mitochondrial F1-ATPases.     

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

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

Threonine 183 and adjacent flexible loop residues in the tryptophan synthase alpha subunit have critical roles in modulating the enzymatic activities of the beta subunit in the alpha 2 beta 2 complex.

This study investigates the catalytic and allosteric roles of a flexible loop in the tryptophan synthase alpha 2 beta 2 complex. This loop connects helix 6 and strand 6 in the alpha subunit, an 8-fold alpha/beta barrel polypeptide. We have engineered three mutations in this disordered loop: a deletion of residues 185-187 and the replacement of threonine 183 by serine (T183S) or by alanine (T183A). Position 183 is a site of an inactivating mutation identified by Yanofsky's group (Yanofsky, C., Drapeau, G. R., Guest, J. R., and Carlton, B. C. (1967) Proc. Natl. Acad. Sci. U.S.A. 57, 296-298). The three engineered alpha subunits form stable, stoichiometric alpha 2 beta 2 complexes with the beta subunit which bind alpha and beta subunit ligands. Although changing threonine 183 to serine has little effect on the enzymatic properties, changing threonine 183 to alanine or deleting residues 185-187 results in a 50-fold reduction in the intrinsic activity of the alpha subunit alone and in the alpha site activity of the alpha 2 beta 2 complex. The latter two mutations profoundly alter the way in which the alpha subunit modulates the spectral properties and the activities of the wild-type beta subunit. These mutations also eliminate the effects of alpha subunit ligands on the beta subunit. Although the beta subunit ligand, L-serine, greatly stabilizes the wild-type alpha 2 beta 2 complex to dissociation and to proteolysis, L-serine stabilizes the T183A alpha 2 beta 2 complex weakly or not at all. Our findings suggest that the hydroxyl residue at position 183 and the adjacent residues in the alpha subunit loop play critical roles in the reciprocal communication between the alpha and beta subunits in the alpha 2 beta 2 complex. The results also help to explain how the wild-type alpha subunit or ammonium ion modulates the activities of the beta subunit.     

5-Fluoroindole Resistance Identifies Tryptophan Synthase Beta Subunit Mutants in Arabidopsis Thaliana

A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation.     

The alpha subunit of tryptophan synthase. Evidence that aspartic acid 60 is a catalytic residue and that the double alteration of residues 175 and 211 in a second-site revertant restores the proper geometry of the substrate binding site

Our studies, which are aimed at understanding the catalytic mechanism of the alpha subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to explore the functional roles of aspartic acid 60, tyrosine 175, and glycine 211. These residues are located close to the substrate binding site of the alpha subunit in the three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Our finding that replacement of aspartic acid 60 by asparagine, alanine, or tyrosine results in complete loss of activity in the reaction catalyzed by the alpha subunit supports a catalytic role for aspartic acid 60. Since the mutant form with glutamic acid at position 60 has partial activity, glutamic acid 60 may serve as an alternative catalytic base. The mutant form in which tyrosine 175 is replaced by phenylalanine has substantial activity; thus the phenolic hydroxyl of tyrosine 175 is not essential for catalysis or substrate binding. Yanofsky and colleagues have identified many missense mutant forms of the alpha subunit of tryptophan synthase from Escherichia coli. Two of these inactive mutant forms had either tyrosine 175 replaced by cysteine or glycine 211 replaced by glutamic acid. Surprisingly, a second-site revertant which contained both of these amino acid changes was partially active. These results indicated that the second mutation must compensate in some way for the first. We now extend the studies of the effects of specific amino acid replacements at positions 175 and 211 by two techniques: 1) characterization of several mutant forms of the alpha subunit from S. typhimurium prepared by site-directed mutagenesis and 2) computer graphics modeling of the substrate binding site of the alpha subunit using the x-ray coordinates of the wild type alpha 2 beta 2 complex from S. typhimurium. We conclude that the restoration of alpha subunit activity in the doubly altered second-site revertant results from restoration of the proper geometry of the substrate binding site.     

Allosteric communication of tryptophan synthase. Functional and regulatory properties of the beta S178P mutant

The alpha(2)beta(2) tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the betaS178P mutant. Ser-178 is located at the end of helix 6 of the beta subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of betaSer-178 is hydrogen bonded to Gly-181 of loop 6 of the alpha subunit only when alpha subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the betaS178P mutation suggests that the hydrogen bond involving alphaGly-181 is disrupted as a result of localized structural perturbations. The ratio of alpha to beta subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight alpha-beta complex. Both the activity of the alpha subunit and the inhibitory effect of the alpha subunit ligands indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the beta subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate do not affect the beta activity. Accordingly, the distribution of l-serine intermediates at the beta-site, dominated by the alpha-aminoacrylate, is only slightly influenced by alpha subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the alpha-aminoacrylate tautomer absorbing at 450 nm. Crystals of the betaS178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the alpha and beta activity in the alpha2beta2 complex with respect to the isolated subunits results from interactions that involve residues different from betaSer-178 and (ii) betaSer-178 is a critical residue in ligand-triggered signals between alpha and beta active sites.     

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.     

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.     

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.     

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.     

Site-directed mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium

Site-specific mutagenesis has been used to prepare two mutant forms of the alpha subunit of tryptophan synthase from Salmonella typhimurium in which either cysteine-81 or cysteine-118 is replaced by a serine residue. These mutant proteins are potentially useful for x-ray crystallographic studies since a heavy metal binding site is specifically eliminated in each mutant. The purified mutant proteins are fully active in four reactions catalyzed by the wild type alpha 2 beta 2 complex of tryptophan synthase. However, the mutant alpha 2 beta 2 complexes dissociate more readily and are less heat-stable than the wild type alpha 2 beta 2 complex. Thus, cysteine-81 and cysteine-118 of the alpha subunit serve structural but not functional roles.     

Escherichia coli tryptophan synthase: synthesis of catalytically competent alpha subunit in a cell-free system containing preacylated tRNAs

A cell-free protein biosynthesizing system prepared from Escherichia coli CF300 was found to synthesize E. coli tryptophan synthase alpha subunit in a time-dependent manner when programmed with pBN69 plasmid DNA. This plasmid contains the trp promoter from Serratia marcescens adjacent to the coding region of E. coli tryptophan synthase alpha protein [Nichols, B.P., & Yanofsky, C. (1983) Methods Enzymol. 101, 155-164]. The synthesized tryptophan synthase alpha subunit was found to be indistinguishable from authentic alpha subunit protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have the same specific activity for catalyzing the conversion of indole----L-tryptophan by tryptophan synthase beta 2 subunit, as well as the conversion of indole + glyceraldehyde 3-phosphate to indole-3-glycerol phosphate. In the absence of exogenously added phenylalanine, admixture of E. coli phenylalanyl-tRNAPhe to the protein biosynthesizing system stimulated the production of functional alpha protein; the analogous result was obtained when valine was replaced by E. coli valyl-tRNAVal. The ability of a misacylated tRNA to participate in alpha protein synthesis in this system was established by the use of E. coli phenylalanyl-tRNAVal in the absence of added valine. Protein biosynthesis proceeded normally and gave a product having the approximate molecular weight of tryptophan synthase alpha subunit; as expected, this polypeptide lacked catalytic activity.     

Subunit stoichiometry of retinal rod cGMP phosphodiesterase

The cyclic GMP phosphodiesterase of the retinal rod is composed of three distinct types of polypeptides: alpha (90 kDa), beta (86 kDa), and gamma (10 kDa). The gamma subunit has been shown to inhibit phosphodiesterase activity associated with alpha and beta. To investigate the subunit stoichiometry of the retinal phosphodiesterase, we have developed a panel of monoclonal and peptide antibodies that recognize individual phosphodiesterase subunits. By quantitative and immunochemical analysis of the purified subunits, we have shown that each phosphodiesterase molecule contains one copy each of alpha and beta subunit and two copies of gamma subunit. Moreover, gamma can be chemically cross-linked to both alpha and beta, but not to itself, suggesting that alpha and beta may each bind one gamma. The phosphodiesterase is fully activated when both copies of gamma were removed by proteolysis with trypsin. Upon recombination of the purified gamma subunit with the trypsin-activated phosphodiesterase containing alpha beta, the alpha beta gamma 2 stoichiometry is once again restored, with concomitant total inhibition of activity. Our results suggest that at least two activated transducin molecules are required to fully activate one molecule of phosphodiesterase in retinal rods.