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E W Miles

Publications and source records attributed to E W Miles.

At least 19 recordsLinked to original sources
PubMed1992

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.

Cloning, Molecular↗
PubMed1992

Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex.

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction. However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., Brzović, P., Drewe, W.F., Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an "open" to a "closed" structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open to the closed form.

Allosteric Regulation↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

Origin of the mutual activation of the alpha and beta 2 subunits in the alpha 2 beta 2 complex of tryptophan synthase. Effect of alanine or glycine substitutions at proline residues in the alpha subunit.

To understand how the alpha and beta 2 subunits of tryptophan synthase from Escherichia coli interact to form an alpha 2 beta 2 complex and undergo mutual activation, we have investigated alpha subunits with single amino acid replacements at conserved proline residues. Although the activities of alpha 2 beta 2 complexes that contain wild type alpha subunit or alpha subunits substituted at positions 28, 62, 96, and 207 are similar, the activities of alpha 2 beta 2 complexes that contain alpha subunits substituted at positions 57 and 132 are remarkably altered. Whereas the latter enzymes have greatly reduced activities in the individual half-reactions, they have considerably higher activities in the overall reaction. These remarkable activity results are explained by a decrease in the affinity of these mutant alpha subunits for the beta 2 subunit and by an increase in the affinity in the combined presence of ligands of both the alpha subunit and the beta 2 subunit. Isothermal calorimetric titrations of wild type beta 2 subunit with wild type alpha subunit and a mutant alpha subunit containing a substitution of glycine for proline at position 132 show that both the affinity and the exothermic association enthalpy are greatly reduced in the mutant alpha subunit although the stoichiometry of association is unchanged. The affinity of the mutant alpha subunit for the beta 2 subunits is greatly increased in the presence of an alpha subunit ligand, alpha-glycerol phosphate. We conclude that proline 132 plays a critical role in subunit interaction and in mutual subunit activation.

Alanine↗
PubMed1992

Subunit communication in the tryptophan synthase alpha 2 beta 2 complex. Effects of beta subunit ligands on proteolytic cleavage of a flexible loop in the alpha subunit.

To probe the structural basis for ligand-mediated communication between the alpha and beta subunits in the tryptophan synthase alpha 2 beta 2 complex, we have determined the effects of ligands of the alpha and beta subunits on proteolysis of a flexible loop in the alpha subunit. We find that addition of a ligand of the beta subunit (L-serine, D-tryptophan, or L-tryptophan) in combination with a ligand of the alpha subunit (alpha-glycerol 3-phosphate) almost completely prevents the tryptic cleavage of the alpha subunit loop. Thus, the binding of a ligand to the beta-site affects the conformation of the alpha subunit 25-30 A distant.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the beta-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium.

In an effort to understand the catalytic mechanism of the tryptophan synthase beta-subunit from Salmonella typhimurium, possible functional active site residues have been identified (on the basis of the 3-D crystal structure of the bienzyme complex) and targeted for analysis utilizing site-directed mutagenesis. The chromophoric properties of the pyridoxal 5'-phosphate cofactor provide a particularly convenient and sensitive spectral probe to directly investigate changes in catalytic events which occur upon modification of the beta-subunit. Substitution of Asp for Glu 109 in the beta-subunit was found to alter both the catalytic activity and the substrate specificity of the beta-reaction. Steady-state kinetic data reveal that the beta-reaction catalyzed by the beta E109D alpha 2 beta 2 mutant enzyme complex is reduced 27-fold compared to the wild-type enzyme. Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy shows that the mutation does not seriously affect the pre-steady-state reaction of the beta E109D mutant with L-serine to form the alpha-aminoacrylate intermediate, E(A-A). Binding of the alpha-subunit specific ligand, alpha-glycerol phosphate (GP) to the alpha 2 beta 2 complex exerts the same allosteric effects on the beta-subunit as observed with the wild-type enzyme. However, the pre-steady-state spectral changes for the reaction of indole with E(A-A) show that the formation of the L-tryptophan quinonoid, E(Q3), is drastically altered. Discrimination against E(Q3) formation is also observed for the binding of L-tryptophan to the mutant alpha 2 beta 2 complex in the reverse reaction. In contrast, substitution of Asp for Glu 109 increases the apparent affinity of the beta E109D alpha-aminoacrylate complex for the indole analogue indoline and results in the increased rate of synthesis of the amino acid product dihydroiso-L-tryptophan. Thus, the mutation affects the covalent bond forming addition reactions and the nucleophile specificity of the beta-reaction catalyzed by the bienzyme complex.

Aspartic Acid↗
PubMed1992

Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium.

To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual alpha and beta subunits. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.

Amino Acid Sequence↗
PubMed1991

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.

Base Sequence↗
PubMed1991

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.

Allosteric Regulation↗
PubMed1991

Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism.

Tryptophan synthase, an alpha 2 beta 2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1991

Site-directed mutagenesis of the beta subunit of tryptophan synthase from Salmonella typhimurium. Role of active site glutamic acid 350.

To investigate the functional role of glutamic acid 350 in the active site of the beta subunit of tryptophan synthase from Salmonella typhimurium, we have replaced this residue by glutamine or alanine by use of site-directed mutagenesis. The mutant alpha 2 beta 2 complexes were expressed, purified, crystallized, and characterized by spectroscopic and kinetic studies with several substrates. We find large alterations in the substrate and reaction specificity of each mutant form of the alpha 2 beta 2 complex. Since the two mutant enzymes are virtually inactive in reactions with L-serine but are active in reactions with beta-chloro-L-alanine, glutamic acid 350 may facilitate the beta-elimination of the weak hydroxyl leaving group of L-serine. The mutant alpha 2 beta 2 complexes are more active than the wild type enzyme in the beta-elimination reaction with beta-chloro-L-alanine. These enzymes are irreversibly inactivated by beta-chloro-L-alanine, whereas the wild type enzyme is not. These altered properties may result from a change in the conformation of the active site, from a change in the orientation of the coenzyme relative to active site residues, or from a change in the solvent accessibility of the active site. The alteration in the active site may enhance the release of amino acrylate from the Schiff base intermediate by hydrolysis or by transamination.

Amino Acids↗
PubMed1991

A colorimetric assay for a pyridoxal phosphate-dependent beta-replacement reaction with L-cysteine: application to studies of wild-type and mutant tryptophan synthase alpha 2 beta 2 complexes.

We present an improved and simple direct assay for formation of inorganic sulfide from L-cysteine in a beta-replacement reaction catalyzed by tryptophan synthase. This method provides a useful enzymatic assay for pyridoxal phosphate-dependent beta-replacement reactions in which the amino acid substrate is L-cysteine and the cosubstrate is 2-mercaptoethanol. The assay should be applicable to similar reactions with L-cysteine and other cosubstrates. The method has several advantages over other methods which have been used to assay similar beta-replacement reactions. The assay is highly reproducible and sensitive and is conveniently carried out in disposable 1.5-ml centrifuge tubes. The color remains stable for several hours. The thiol compounds L-cysteine and 2-mercaptoethanol do not interfere at the concentrations used. The method has useful applications to studies of the rates and reaction specificities of several other pyridoxal phosphate enzymes which catalyze beta-replacement reactions. We demonstrate the use of the method to study the effects of site-directed mutagenesis on the reaction specificity and mechanism of the tryptophan synthase alpha 2 beta 2 complex.

Colorimetry↗
PubMed1991

Effect of single amino acid substitutions at positions 49 and 60 on the thermal unfolding of the tryptophan synthase alpha subunit from Salmonella typhimurium.

We have used circular dichroism measurements to compare the thermal unfolding of the wild type tryptophan synthase alpha subunit from Salmonella typhimurium with that of seven mutant forms with single amino acid replacements at two active site residues. Glutamic acid 49 has been replaced by phenylalanine, glutamine, or aspartic acid. Aspartic acid 60 has been replaced by alanine, aspartic acid, asparagine, or tyrosine. Thermodynamic properties (delta G, delta H, delta S, and Tm) of the wild type and mutant forms have been determined experimentally by measuring the free energy of unfolding as a function of temperature. Increasing the pH from 7.0 to 8.8 decreases the tm of the wild type alpha subunit from 56 to 45 degrees C. The thermal unfolding of the wild type alpha subunit and of six of the seven mutant forms can be described as reversible, two-state transitions. In contrast, the melting curve of a mutant alpha subunit in which aspartic acid 60 is replaced by tyrosine indicates the presence of a folding intermediate which may correspond to a "molten globule." Correlations between our observations and previous folding studies and the X-ray crystallographic structure are presented. Substitution of glutamic acid 49, which is located in the hydrophobic "pit" of an eight-fold alpha/beta barrel, by a hydrophobic phenylalanine residue increases the tm from 56 to 60 degrees C. In contrast, replacement of aspartic acid 60, which is accessible to solvent, results in small reductions in the thermal stability.

Circular Dichroism↗
PubMed1990

The tryptophan synthase multienzyme complex: exploring structure-function relationships with X-ray crystallography and mutagenesis.

The bifunctional tryptophan synthase alpha 2 beta 2 complex that catalyzes the final two reactions in tryptophan biosynthesis is a classic example of a multienzyme complex that "channels" a metabolic intermediate (indole) between two active sites. The three-dimensional structure of the alpha 2 beta 2 complex from Salmonella typhimurium reveals that the four polypeptide subunits are arranged in an extended alpha beta beta alpha order forming a complex 150 A long. The active sites of the neighboring alpha and beta subunits are separated by about 30 A and appear to be connected by a tunnel, which may facilitate the intramolecular transfer of indole. The active site of the alpha subunit, which is centrally located near one end of an eight-fold alpha/beta barrel structure, contains the sites of most of the missense mutations which were identified as key residues by Yanofsky and colleagues in early genetic studies. Site-directed mutagenesis is being used to replace residues found in the active sites of the alpha and beta subunits in order to probe the mechanism of catalysis. Recombinant DNA technology should also be useful in analyzing protein-protein interaction, protein folding and the channeling phenomenon.

Crystallography↗
PubMed1989

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

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

Crystallization↗
PubMed1989

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.

Arginine↗
PubMed1989

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.

Aspartic Acid↗