Structural and functional influence of enzyme-antibody interactions: effects of eight different monoclonal antibodies on the enzymatic activity of Escherichia coli tryptophan synthase

Twelve monoclonal antibodies directed against the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were produced from hybridoma clones. These monoclonal antibodies are found to recognize at least eight different epitopes on beta 2, and eight classes of monoclonal antibodies are thus defined. The effects of these monoclonal antibodies on the enzymatic activities of beta 2 are studied. The monoclonal antibodies from three classes rapidly inhibit the serine deaminase activity catalyzed by the beta 2 subunit alone; two of them lead to an inhibition plateau under stoichiometric conditions, and their inhibitory effects are cumulative. With the antibodies from two of these three classes, the tryptophan synthase activity of the alpha 2 beta 2 complex is recovered, through a competition between the alpha subunit and the monoclonal antibody. On the contrary, the antibody from the third class is inhibitory even in the presence of an excess of alpha subunit. The antibodies from the five other classes, though binding easily to the coated antigen in the enzyme-linked immunosorbent assay, react only very slowly with beta 2 in solution and, only after a long time of incubation, inhibit the enzymatic activity at different levels.     

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.     

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.     

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.     

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.     

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.     

The beta subunit of tryptophan synthase. Clarification of the roles of histidine 86, lysine 87, arginine 148, cysteine 170, and cysteine 230

Our studies, which are aimed at understanding the catalytic mechanism of the beta subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to clarify the functional roles of several putative active site residues. Although previous chemical modification studies have suggested that histidine 86, arginine 148, and cysteine 230 are essential residues in the beta subunit, our present findings that beta subunits with single amino acid replacements at these positions have partial activity show that these 3 residues are not essential for catalysis or substrate binding. These conclusions are consistent with the recently determined three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Amino acid substitution of lysine 87, which forms a Schiff base with pyridoxal phosphate in the wild type beta subunit, yields an inactive form of the beta subunit which binds alpha subunit, pyridoxal phosphate, and L-serine. We also report a rapid and efficient method for purifying wild type and mutant forms of the alpha 2 beta 2 complex from S. typhimurium from an improved enzyme source. The enzyme, which is produced by a multicopy plasmid encoding the trpA and trpB genes of S. typhimurium expressed in Escherichia coli, is crystallized from crude extracts by the addition of 6% poly(ethylene glycol) 8000 and 5 mM spermine. This new method is also used in the accompanying paper to purify nine alpha 2 beta 2 complexes containing mutant forms of the alpha subunit.     

Regulation of tryptophan synthase by temperature, monovalent cations, and an allosteric ligand. Evidence from Arrhenius plots, absorption spectra, and primary kinetic isotope effects

To investigate the linkage between enzyme conformation and catalysis, we have determined the effects of temperature on catalytic properties of the tryptophan synthase alpha(2)beta(2) complex and beta(2) subunit in the absence or presence of different monovalent cations (Cs(+), Na(+), and GuH(+)) and of an allosteric ligand, alpha-glycerol 3-phosphate. Arrhenius plots of the activity data between 5 and 50 degrees C are nonlinear in the presence of certain ligands but not others. The conditions that yield nonlinear Arrhenius plots also yield temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. The results provide evidence that the nonlinear Arrhenius plots are caused by a temperature-dependent conformational change that precedes the rate-limiting step in catalysis. Thermodynamic analysis of the data associated with the conformational change shows that the activation energies are much higher at low temperatures than at high temperatures. We correlate the results with a model in which the enzyme is converted by increased temperature under certain conditions from a low-activity "open" conformation to a high-activity "closed" conformation. The allosteric ligand and different monovalent cations, including GuH(+), which also acts as a chaotropic agent, affect the equilibrium between the open and closed forms. The large positive entropy changes in the conformational conversion suggest that the closed conformation results from tightened hydrophobic interactions that exclude water from the active site of the beta subunit.     

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.     

Genetic and biochemical characterization of the trpB8 mutation of Escherichia coli tryptophan synthase. An amino acid switch at the sharp turn of the trypsin-sensitive "hinge" region diminishes substrate binding and alters solubility.

The trpB8 mutation of Escherichia coli tryptophan synthase is unique in that the cells bearing this lesion are not only capable of utilizing indole for growth, but they also accumulate indole, under conditions of tryptophan limitation. The lesion was shown by DNA sequencing to be a G to C transversion at nucleotide 5528 of the trp operon, resulting in a Gly to Arg switch at codon 281. Gly-281, within the trypsin-sensitive "hinge" region, is invariant among all known beta polypeptides. The catalytic activity of the mutant beta 2(B8) protein is dramatically stimulated by alpha subunit, both in vivo and in vitro. In the absence of alpha subunit, ammonium ion effectively stimulated the activity in an apparently cooperative manner. The pH optimum for the mutant subunit was 9.8, which is 2 units higher than that of wild type. In contrast to the wild-type subunit, beta(B8) partially aggregated within cells upon overexpression. At the optimal concentration of ammonium ions (2.25 M), the beta 2(B8) mutant enzyme displayed lower affinity than wild-type enzyme toward indole and L-serine, but the Vmax was almost unchanged. The physicochemical behavior of beta 2(B8) is supported by computer graphic modeling studies. An open versus closed model of conformational change within the beta 2 protein is proposed. A plausible role for the hinge region is discussed.     

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.     

Mechanism of inactivation of the beta 2 subunit of Escherichia coli tryptophan synthase by monoclonal antibodies.

Monoclonal antibodies directed against the native form of the beta 2 subunit of Escherichia coli tryptophan synthase strongly inhibit both its tryptophan synthase and its serine deaminase activities. The mechanism of this inactivation is studied here, by monitoring quantitatively the absorption and fluorescence properties of different well-characterized successive intermediates in the catalytic cycle of tryptophan synthase. It is shown that the antibodies interfere specifically with the formation of one or the other of these intermediates. It is concluded that the antibodies either modify or block the molecular flexibility of the protein, thus preventing conformational changes that the protein has to undergo during the catalysis. At least two different stages of the catalytic process, each one sensitive to a different class of antibodies, are shown to involve molecular movements of the polypeptide chain. Indications are given on the regions of the molecule involved in these movements.     

Fluorescence-quenching studies on a conformational transition within a domain of the beta 2 subunit of Escherichia coli tryptophan synthase

The fluorescence quenching by acrylamide of the single tryptophan residue in the beta 2 subunit of tryptophan synthase from Escherichia coli K12 is studied for different states of the protein: the native apo-enzyme and holo-enzyme, the nicked apo-protein and holo-protein and the isolated proteolytic fragment F1 corresponding to the N-terminal two thirds of beta 2. The quenching constants measured are used to estimate the accessibility of the tryptophan residue in these different forms. The results are discussed in terms of conformational transition within the F1 domain, occurring in the presence of the cofactor, pyridoxal 5'-phosphate, in the native enzyme. The proteolytic cleavage of the native enzyme is shown to render the nicked protein unable to undergo this conformational change.     

Monoclonal antibodies as probes of the topological arrangement of the alpha subunits of Escherichia coli RNA polymerase

Three monoclonal anti-alpha antibodies were used to study the properties of the alpha subunit of Escherichia coli RNA polymerase. None of the monoclonal antibodies inhibited the d(A-T)n-directed synthesis of r(A-U)n. Reassembly of the RNA polymerase core was blocked by mAb 129C4 or mAb 126C6 while no effect was observed with mAb 124D1. The conversion of premature to mature core was partially inhibited by mAb 129C4 and almost totally inhibited by mAb 126C6. The data suggest that during the course of core assembly at least one of the alpha subunits undergoes conformational changes. The increase in affinity of mAb 126C6 for assembled alpha compared with free alpha also implies that alpha undergoes conformational changes during RNA polymerase assembly. Double antibody binding studies showed that the epitopes for mAb 124D1 and mAb 129C4 are available on only one of the alpha subunits in RNA polymerase. It would appear that the relevant domain on one of the alpha subunits in RNA polymerase is well exposed whereas this domain on the second alpha subunit is shielded by interaction with regions of the large beta and beta' subunits. The alpha domain in which the epitope for mAb 126C6 resides is not impeded by subunit interactions in the RNA polymerase. The data obtained also suggest that in the holoenzyme the sigma subunit may be positioned close to one of the alpha subunits, probably to the more exposed alpha. The alpha beta complex is the minimal stable subassembly since one of the alpha subunits dissociates from the alpha 2 beta complex following binding of any of the monoclonal antibodies studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylase kinase conformers. Detection by proteases

A variety of proteases have been evaluated as potential structural and conformational probes of nonphosphorylated and phosphorylated phosphorylase kinase. In general, the enzyme's alpha subunit is rapidly degraded, followed in most cases by hydrolysis of the beta subunit; the gamma subunit is resistant to most proteases. Trypsin clearly distinguishes between the nonactivated and activated conformers of phosphorylase kinase, in that the beta subunit in phosphorylated enzyme, as opposed to nonphosphorylated enzyme, is markedly protected from tryptic attack. In contrast, only a small difference in the rates of proteolysis of the alpha subunit in phosphorylated and nonphosphorylated enzyme is seen, even when a protease is used that is highly selective for the alpha subunit, such as chymotrypsin or endoproteinase Arg C. Incubation of nonphosphorylated phosphorylase kinase with either Mg2+ or Ca2+, which are activating cations, also protects the beta subunit from tryptic hydrolysis, whereas Mn2+, which inhibits the kinase activity, has little effect on proteolysis. The allosteric activator ADP also causes the beta subunit to become refractory to trypsin and mimics the effects of phosphorylation. Similar effector-induced conformational changes in the beta subunit are also observed with enzyme in which the alpha subunit has previously been selectively destroyed. These data indicate that activation of phosphorylase kinase by dissimilar mechanisms is associated with a conformational change in the enzyme's beta subunit that is detectable by trypsin and confirm earlier studies from this laboratory employing a chemical cross-linker as a conformational probe for activated and nonactivated conformers of the enzyme (Fitzgerald, T. J., and Carlson, G. M. (1984) J. Biol. Chem. 259, 3266-3274).     

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.     

An active alpha'2beta2 derivative of tryptophean synthase formed by limited proteolysis.

A new approach to studying the arrangement of subunits in the multienzyme complex tryptophan synthase is reported. Comparative studies of limited tryptic proteolysis of the alpha2beta2 complex and of the separate beta2 and alpha subunits show that subunit association inhibits two types of proteolysis which occur with the separate subunits: (i) cleavage of the beta2 subunit to two fragments with consequent loss of activity and (ii) complete degradation of the alpha subunit with loss of activity. Trypsin treatment of the alpha2beta complex does, however, result in at least one cleavage of the alpha subunit and yields an active alpha'2beta2 complex. The alpha'2beta2 complex can be resolved into an active beta2 subunit and an active alpha derivative termed alpha'. These two species can reassociate into the active alpha'2beta2 complex. alpha' derivative can be separated into a large fragment of Mr approximately 20,000 to 23,000 and a small peptide by polyacrylamide gel electrophoresis under denaturing conditions.     

Evidence for two conformers of the beta subunit of tryptophan synthase in solution.

To explain our finding that the dimeric beta subunit of tryptophan synthase is only 50% inactivated by beta-chloro-L-alanine (Ahmed, S. A., Ruvinov, S. B., Kayastha, A. M., and Miles, E. W. (1991) J. Biol. Chem. 266, 21548-21557), we have extended our investigation using spectroscopic, steady-state kinetic, and electrophoretic methods. The spectroscopic properties of the half-active beta 2 dimer and the reactivation after alkali treatment show that the inactivation proceeds by an "enamine" mechanism. Although the fully active beta 2 dimer associates with the tryptophan synthase alpha subunit to form alpha 2 beta 2 complex, the inactive beta subunits in the half-active enzyme associate weakly or not at all with the alpha subunit. Our results provide evidence for two conformers of the beta subunit in solution: one is rapidly inactivated by beta-chloro-L-alanine and the other is not inactivated. Thermal inactivation studies and non-denaturing polyacrylamide gel electrophoresis of the half-active enzyme show that the beta 2 dimer exists in both homologous and heterologous combinations of these two forms. After removal of the reaction products and unreacted beta-chloro-L-alanine from the half-active beta 2 dimer by gel filtration, further incubation with beta-chloro-L-alanine results in the loss of 50% of the remaining activity. This result suggests that the subunits undergo rearrangement via an intermediate monomer form to regenerate the two conformers of the active beta subunit. This mechanism of rearrangement is supported by our finding that the extent of inactivation increases at lower concentrations of the beta 2 dimer.