Kinetic studies on the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes.

Steady-state kinetic techniques have been used to investigate each of the reactions catalyzed by the bifunctional enzyme, chorismate mutase-prephenate dehydrogenase, from Aerobacter aerogenes. The results of steady-state velocity studies in the absence of products, as well as product and dead-end inhibition studies, suggest that the prephenate dehydrogenase reaction conforms to a rapid equilibrium random mechanism which involes the formation of two dead-end complexes, viz, enzyme-NADH-prephenate and enzyme-NAD+-hydroxyphenylpyruvate. Chorismate functions as an activator of the dehydrogenase while both prephenate and hydroxyphenylpyruvate acted as competitive inhibitors in the mutase reaction. By contrast. bpth NAD+ and NADH function as activators of the mutase. Values of the kinetic parameters associated with the mutase and dehydrogenase reactions have been determined and the results discussed in terms of possible relationships between the catalytic sites for the two reactions. The data appear to be consistent with the enzyme having either a single site at which both reactions occur or two separate sites which possess similar kinetic properties.     

Kinetic studies on chorismate mutase-prephenate dehydrogenase from Escherichia coli: models for the feedback inhibition of prephenate dehydrogenase by L-tyrosine

Kinetic studies have been undertaken to elucidate the mechanism of the allosteric inhibition by tyrosine of the prephenate dehydrogenase activity of the bifunctional dimeric enzyme chorismate mutase-prephenate dehydrogenase. The effect of tyrosine on the initial velocity of the reactions in the presence of both prephenate and the alternative substrate, 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate, have been determined. In addition, investigations have been made of the effect of tyrosine on the inhibition of the reaction by the inhibitory analogues of prephenate, (4-hydroxyphenyl)pyruvate, and (carboxyethyl)-1,4-dihydrobenzoate. The results of the double inhibition experiments indicate clearly that the enzyme possesses a distinct allosteric site for the binding of tyrosine. The initial velocity data obtained with both substrates have been fitted to the rate equations that describe a wide range of models. From a comparison of the results obtained from studies with the two substrates, and with a knowledge of the value for the dissociation constant of the tyrosine-enzyme complex, definitive conclusions have been reached about the mechanism of the allosteric inhibition. It is concluded that tyrosine combines twice at allosteric sites and in an antisynergistic fashion, while prephenate reacts at both active sites of the dimeric enzyme as well as weakly at one of the allosteric sites. It appears that the latter is simple competition reaction that affects neither the binding of prephenate at the active site nor the rate of product formation. The model also predicts the formation of an active tyrosine-enzyme-prephenate complex that yields product at a much slower rate than does the enzyme-prephenate complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: cooperative effects and inhibition by L-tyrosine

The effects of a variety of structural analogs of L-tyrosine on the mutase and dehydrogenase activities of hydroxyphenylpyruvate synthase have been investigated. From these studies it is concluded that the alpha-NH3+ alpha-COO-, and the 4-OH groups are essential for binding of L-tyrosine as an inhibitor of the dehydrogenase and that the L configuration is also essential. Dixon plots for inhibition of the dehydrogenase activity by some of these analogs were nonlinear and could be described by a velocity equation that is the ratio of quadratic polynomials (a 2/1 function). Dixon plots for inhibition of the mutase by prephenate at low concentrations of chorismate could also be described by a 2/1 function, but at low concentrations of prephenate chorismate acts as an apparent hyperbolic activator of the dehydrogenase activity. Up to concentrations of 300 microM, L-tyrosine activates the mutase but acts as a potent inhibitor of the dehydrogenase. Such data for the dehydrogenase could not be described by a 2/1 function in 1/[prephenate] but could be fitted to the Hill equation with increasing concentrations of L-tyrosine in the presence of 1.0 mM NAD yielding increasing values for the Hill number (n): in the absence of L-tyrosine, n = 1.6 +/- 0.1; at 150 microM L-tyrosine, n = 2.1 +/- 0.1; at 300 microM L-tyrosine, n = 2.3 +/- 0.4. L-Tyrosine bears a close structural resemblance to both prephenate and hydroxyphenylpyruvate, and evidence is presented which is consistent with L-tyrosine acting as a competitive inhibitor with respect to prephenate of the dehydrogenase.     

Metabolic engineering of Escherichia coli for L-tyrosine production by expression of genes coding for the chorismate mutase domain of the native chorismate mutase-prephenate dehydratase and a cyclohexadienyl dehydrogenase from Zymomonas mobilis

The expression of the feedback inhibition-insensitive enzyme cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase domain from native chorismate mutase-prephenate dehydratase (PheA(CM)) from Escherichia coli was compared to the expression of native feedback inhibition-sensitive chorismate mutase-prephenate dehydrogenase (CM-TyrA(p)) with regard to the capacity to produce l-tyrosine in E. coli strains modified to increase the carbon flow to chorismate. Shake flask experiments showed that TyrC increased the yield of l-tyrosine from glucose (Y(l-Tyr/Glc)) by 6.8-fold compared to the yield obtained with CM-TyrA(p). In bioreactor experiments, a strain expressing both TyrC and PheA(CM) produced 3 g/liter of l-tyrosine with a Y(l-Tyr/Glc) of 66 mg/g. These values are 46 and 48% higher than the values for a strain expressing only TyrC. The results show that the feedback inhibition-insensitive enzymes can be employed for strain development as part of a metabolic engineering strategy for l-tyrosine production.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: spatial relationship of the mutase and dehydrogenase sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (4-hydroxyphenylpyruvate synthase) by substrate analogues has been investigated at pH 6.0 with the aim of elucidating the spatial relationship that exists between the sites at which each reaction occurs. Several chorismate and adamantane derivatives, as well as 2-hydroxyphenyl acetate and diethyl malonate, act as linear competitive inhibitors with respect to chorismate in the mutase reaction and with respect to chorismate in the mutase reaction and with respect to prephenate in the dehydrogenase reaction. The similarity of the dissociation constants for the interaction of these compounds with the free enzyme, as determined from the mutase and dehydrogenase reactions, indicates that the reaction of these inhibitors at a single site prevents the binding of both chorismate and prephenate. However, not all the groups on the enzyme, which are responsible for the binding of these two substrates, can be identical. At lower concentrations, citrate or malonate prevents reaction of the enzyme with prephenate, but not with chorismate. Nevertheless, the combining sites for chorismate and prephenate are in such close proximity that the diethyl derivative of malonate prevents the binding of both substrates. The results lead to the proposal that the sites at which chorismate and prephenate react on hydroxyphenylpyruvate synthase share common features and can be considered to overlap.     

Production of chorismate mutase-prephenate dehydrogenase by a strain of Escherichia coli carrying a multicopy,tyrA plasmid: Isolation and properties of the enzyme

A multicopy plasmid that contains the tyrosine operon has been used to transform strains of Escherichia coli K-12. The resultant strains yielded levels of chorismate mutase-prephenate dehydrogenase that were up to 5000-fold higher than that given by the parent strain and about 6-fold higher than that given by a tyrR strain. The production of enzyme fell when tetracycline was omitted from the growth medium because of the loss of the plasmid. The bifunctional enzyme was isolated in good yield by a simple purification procedure and shown to possess properties identical to those exhibited by the enzyme from a tyrR strain.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: positive cooperativity with substrates and inhibitors

Investigations have been made at pH 6.0 of the effect of chorismate and adamantane derivatives on the mutase and dehydrogenase activities of hydroxyphenylpyruvate synthase from Escherichia coli. When used over a wide range of concentrations, chorismate 5,6-epoxide, chorismate 5,6-diol, adamantane-1,3-diacetate, adamantane-1-acetate, adamantane-1-carboxylate, and adamantane-1-phosphonate give rise to nonlinear plots of the reciprocal of the initial velocity of each reaction as a function of the inhibitor concentration. The inhibitors do not induce the enzyme to undergo polymerization and have only a small effect on the S20,w value of the enzyme as determined by using sucrose density gradient centrifugation. At low substrate concentration, low concentrations of adamantane-1-acetate cause activation of both the mutase and dehydrogenase activities while at higher concentrations this compound functions as an inhibitor. When chorismate and prephenate are varied over a wide range of concentrations, double-reciprocal plots of the data indicate that the reactions exhibit positive cooperativity. The addition of albumin eliminates the cooperative interactions associated with substrates but has little effect on those associated with inhibitors.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 1. Kinetic characterization of the dehydrogenase reaction by use of alternative substrates

The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase-prephenate dehydrogenase, has been isolated from extracts of a plasmid-containing strain of Escherichia coli K12 and purified to homogeneity by a modified procedure that involves chromatography on both Matrex Blue A and Sepharose-AMP. Detailed studies of the dehydrogenase reaction have been undertaken with analogues of prephenate that act as substrates. The analogues, which included two of the four possible diastereoisomers of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate (deoxodihydroprephenate) as well as D- and L-arogenate, were synthesized chemically. As judged by their V/K values, all analogues were poorer substrates than prephenate. The order of their effectiveness as substrates is prephenate greater than one isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than L-arogenate greater than other isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than D-arogenate. Thus the dehydrogenase activity is dependent on the degree and position of unsaturation in the ring structure of prephenate as well as on the type of substitution on the pyruvyl side chain. With prephenate as a substrate, the reaction is irreversible because it involves oxidative decarboxylation. By contrast, 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate undergoes only a simple oxidation, and thus, with this substrate, the reaction is reversible. Steady-state velocity data, obtained by varying substrates over a range of higher concentrations, suggest that the dehydrogenase reaction conforms to a rapid equilibrium, random mechanism with 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate as a substrate in the forward reaction or with the corresponding ketone derivative as a substrate in the reverse direction. The initial velocity patterns obtained by varying prephenate or 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate over a range of lower concentrations, at different fixed concentrations of NAD, were nonlinear and consistent with a unique model that is described by a velocity equation which is the ratio of quadratic polynomials. An equilibrium constant of 1.4 x 10(-7) M for the reaction in the presence of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate indicates that the equilibrium lies very much in favor of ketone production.     

Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus.

Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 47,000, indicating a tetramer of identical subunits. The isoelectric point of the bifunctional enzyme was 5.8. Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutasedehydratase by DEAE-cellulose chromatgraphy. Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The molecular weight of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis. Its isoelectric point was pH 6.6. In the overall conversion of chorismate to phenylpyruvate, free prephenate was formed which accumulated in the reaction mixture. The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.     

Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine.

Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo.     

Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein.

The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the l-tyrosine biosynthetic pathway. The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine. Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains. Residues 1-88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback-inhibition activities were localized in residues 94-373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.     

Interconvertible molecular-weight forms of the bifunctional chorismate mutase-prephenate dehydratase from Acinetobacter calcoaceticus

Acinetobacter calcoaceticus belongs to a large phylogenetic cluster of gram-negative procaryotes that all utilize a bifunctional P-protein (chorismate mutase-prephenate dehydratase) [EC 5.4.99.5-4.2.1.51] for phenylalanine biosynthesis. These two enzyme activities from Ac. calcoaceticus were inseparable by gel-filtration or DEAE-cellulose chromatography. The molecular weight of the P-protein in the absence of effectors was 65,000. In the presence of L-tyrosine (dehydratase activator) or L-phenylalanine (inhibitor of both P-protein activities), the molecular weight increased to 122,000. Maximal activation (23-fold) of prephenate dehydratase was achieved at 0.85 mM L-tyrosine. Under these conditions, dehydratase activity exhibited a hysteretic response to increasing protein concentration. Substrate saturation curves for prephenate dehydratase were hyperbolic at L-tyrosine concentrations sufficient to give maximal activation (yielding a Km,app of 0.52 mM for prephenate), whereas at lower L-tyrosine concentrations the curves were sigmoidal. Dehydratase activity was inhibited by L-phenylalanine, and exhibited cooperative interactions for inhibitor binding. A Hill plot yielded an n' value of 3.1. Double-reciprocal plots of substrate saturation data obtained in the presence of L-phenylalanine indicated cooperative interactions for prephenate in the presence of inhibitor. The n values obtained were 1.4 and 3.0 in the absence or presence of 0.3 mM L-phenylalanine, respectively. The hysteretic response of chorismate mutase activity to increasing enzyme concentration was less dramatic than that of prephenate dehydratase. A Km,app for chorismate of 0.63 mM was obtained. L-Tyrosine did not affect chorismate mutase activity, but mutase activity was inhibited both by L-phenylalanine and by prephenate. Interpretations are given about the physiological significance of the overall pattern of allosteric control of the P-protein, and the relationship between this control and the effector-induced molecular-weight transitions. The properties of the P-protein in Acinetobacter are considered within the context of the ubiquity of the P-protein within the phylogenetic cluster to which this genus belongs.     

Stereochemistry and mechanism of reactions catalyzed by tryptophanase Escherichia coli.

Several beta replacement and alpha,beta elimination reactions catalyzed by tryptophanase from Escherichia coli are shown to proceed stereospecifically with retention of configuration. These conversions include synthesis of tryptophan from (2S,3R)- and (2s,3s)-[3(-3H)]serine in the presence of indole, deamination of these serines in D2O to pyruvate and ammonia, and cleavage of (2S,3R)-and (2S,3S)-[3(-3H)]tryptophan in D2O to indole, pyruvate, and ammonia. A coupled reaction with lactate dehydrogenase was used to trap the stereospecifically labeled [3-H,2H,3H]pryuvates as lactate, which was oxidized to acetate for chirality analysis of the methyl group. During deamination of tryptophan there is significant intramolecular transfer of the alpha proton of the amino acid to C-3 of indole. To determine the exposed face of the cofactor.substrate complex on the enzyme surface and to analyze its conformational orientation, sodium boro[3H]hydride was used to reduce tryptophanase-bound alaninepyridoxal phosphate Schiff's base. Degradation of the resulting pyridoxylalanine to (2S)-[2(-3H)]alanine and (4'S)-[4'(-3H)]pyridoxamine demonstrates that reduction occurs from the exposed si face at C-4' of the complex and that the ketimine double bond is trans.     

Identification of an essential cysteine in the reaction catalyzed by aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The enzyme L-aspartate-beta-semialdehyde dehydrogenase from Escherichia coli has been studied by oligonucleotide-directed mutagenesis. The focus of this investigation was to examine the role of a cysteine residue that had been previously identified by chemical modification with an active site directed reagent (Biellmann et al. (1980) Eur. J. Biochem. 104, 59-64). Substitution of this cysteine at position 135 with an alanine results in complete loss of enzyme activity. However, changing this cysteine to a serine yields a mutant enzyme with a maximum velocity that is 0.3% that of the native enzyme. This C135S mutant has retained essentially the same affinity for substrates as the native enzyme, and the same overall conformation as reflected in identical behavior on gel electrophoresis and in identical fluorescence spectra. The pH profile of the native enzyme shows a loss in catalytic activity upon protonation of a group with a pKa value of 7.7. The same activity loss is observed at this pH with the serine-135 mutant, despite the differences in the pKa values for a cysteine sulfhydryl and a serine hydroxyl group that have been measured in model compounds. This observed pKa value may reflect the protonation of an auxiliary catalyst that enhances the reactivity of the active site cysteine nucleophile in the native aspartate-beta-semialdehyde dehydrogenase.