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 dehydratase from Escherichia coli: active sites of a bifunctional enzyme.

The relationship between the active sites of the bifunctional enzyme chorismate mutase-prephenate dehydratase has been examined. Steady-state kinetic investigations of the reactions with chorismate or prephenate as substrate and studies of the overall conversion of chorismate to phenylpyruvate indicate that there are two distinct active sites. One site is responsible for the mutase activity and the other for the dehydratase activity. Studies of the overall reaction using radioactive chorismate show that prephenate, which is formed from chorismate, dissociates from the mutase site and equilibrates with the bulk medium before combining at the dehydratase site. No evidence was obtained for direct channeling of prephenate from one site to the other, or for any strong interaction between the sites.     

Chorismate mutase:prephenate dehydratase from Acinetobacter calcoaceticus. Purification, properties and immunological cross-reactivity

The bifunctional P protein (chorismate mutase: prephenate dehydratase) from Acinetobacter calcoaceticus has been purified. It was homogeneous in polyacrylamide gels and was more than 95% pure on the basis of the immunostaining of purified P protein with the antibodies raised against the P protein. The native enzyme is a homodimer (Mr = 91,000) composed of 45-kDa subunits. A twofold increase in the native molecular mass of the P protein occurred in the presence of L-phenylalanine (inhibitor of both activities) or L-tyrosine (activator of the dehydratase activity) during gel filtration. Chorismate mutase activity followed Michaelis-Menten kinetics with a Km of 0.55 mM for chorismate. L-Phenylalanine was a relatively poor non-competitive inhibitor of the mutase activity. The chorismate mutase activity was also competitively inhibited by prephenate (reaction product). Substrate-saturation curves for the dehydratase activity were sigmoidal showing positive cooperativity among the prephenate-binding sites. L-Tyrosine activated prephenate dehydratase strongly but did not abolish positive cooperativity with respect to prephenate. L-Phenylalanine inhibited the dehydratase activity, and the substrate-saturation curves became increasingly sigmoidal as phenylalanine concentrations were increased with happ values changing from 2.0 (no phenylalanine) to 4.0 (0.08 mM L-phenylalanine). A sigmoidal inhibition curve of the dehydratase activity by L-phenylalanine gave Hill plots having a slope of -2.9. Higher ionic strength increased the dehydratase activity by reducing the positive cooperative binding of prephenate, and the sigmoidal substrate-saturation curves were changed to near-hyperbolic form. The happ values decreased with increase in ionic strength. Antibodies raised against the purified P protein showed cross-reactivity with the P proteins from near phylogenetic relatives of A. calcoaceticus. At a greater phylogenetic distance, cross-reaction was superior with P protein from Neisseria gonorrhoeae than with that from the more closely related Escherichia coli.     

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.     

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.     

Chorismate mutase/prephenate dehydratase from Escherichia coli K12. Binding studies with the allosteric effector phenylalanine.

The binding of phenylalanine to the allosteric site of chorismate mutase/prephenate dehydratase has been studied by steady-state dialysis. Under most of the experimental conditions examined positive co-operativity was observed for the binding of ligand up to 50% saturation and negative co-operativity above 50% saturation. In the presence of 0.4 M NaCl at pH 8.2 the co-operativity was positive at all phenylalanine concentrations and the maximal stoichiometry of 1 mol of phenylalanine/mol of enzyme subunit was observed. It was concluded that there is a single phenylalanine-binding site per subunit which is associated with the regulation of each of the mutase and dehydratase activities. The effects of enzyme concentration, NaCl, temperature and pH on the binding of phenylalanine have been investigated. Neither tyrosine nor tryptophan bound to the allosteric site of the enzyme. Enzyme that was desensitized to inhibition by phenylalanine following modification of three sulphydryl groups with 5,5'-dithio-bis (2-nitrobenzoic acid) did not bind phenylalanine. The mechanism of co-operativity, the binding of the enzyme to Sepharosyl-phenylalanine and the physiological significance of the inhibition of the enzyme by phenylalanine are discussed in terms of the results obtained.     

Chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes: evidence that the two reactions occur at one active site.

The relationship between the sites for catalysis of two reactions by the bifunctional enzyme chorismate mutase--prephenate dehydrogenase has been investigated. The results are consistent with the occurrence of both reactions at one active site. Comparisons have been made between experimental data for the time course of the overall reaction and computer simulations, according to various models for the relationship between the mutase and dehydrogenase sites. A model based on a single active site is consistent with the time course data if a minor proportion of the chorismate that reacts can be converted through to (hydroxyphenyl)pyruvate without the intermediate release of prephenate. Consistent with this requirement, some channeling of radioactivity from chorismate to (hydroxyphenyl)pyruvate has been detected. A model based on two separate sites has also been considered; the simulations show that if this model applies there is no need to postulate any channeling of the intermediate, prephenate, between the sites and there must be marked inhibition of the dehydrogenase reaction by chorismate. Since channeling has been observed and chorismate increases the dehydrogenase rate under all conditions, the two-site model appears unlikely. Consistent with the one-site model are the observations that a variety of inactivating conditions cause parallel loss of mutase and dehydrogenase activity and that identical protection against inactivation of both mutase and dehydrogenase by iodoacetamide is afforded by prephenate.     

Chorismate mutase/prephenate dehydratase from Escherichia coli K12. Effect of phenylalanine, NaCl and pH on the protein conformation.

The effects of phenylalanine, NaCl and pH on the conformation of chorismate mutase/prephenate dehydratase have been investigated, using measurements of far and near-ultraviolet circular dichroic spectra and ultraviolet difference spectra. At pH 8.2 in 20 mM Tris-Cl buffer the enzyme was found to contain 10-20% helix and 40-50% beta-structure. There was little or no change in these values on the addition of 1 mM phenylalanine (the allosteric effector) or 0.4 M NaCl or by decreasing the pH to 7.4. Both phenylalanine and NaCl caused significant changes in the conformation of the enzyme. The most prominent of these was the movement of a tryptophan residue into a more hydrophobic environment. There was also a slight perturbation of this tryptophan when the pH was decreased to 7.4. The conformational changes can explain sigmoidal kinetic behaviour observed previously [Gething et al. (1976) Eur. J. Biochem. 71, 317-325].     

X-ray structure of prephenate dehydratase from Streptococcus mutans

Prephenate dehydratase is a key enzyme of the biosynthesis of L-phenylalanine in the organisms that utilize shikimate pathway. Since this enzymatic pathway does not exist in mammals, prephenate dehydratase can provide a new drug targets for antibiotics or herbicide. Prephenate dehydratase is an allosteric enzyme regulated by its end product. The enzyme composed of two domains, catalytic PDT domain located near the N-terminal and regulatory ACT domain located near the C-terminal. The allosteric enzyme is suggested to have two different conformations. When the regulatory molecule, phenylalanine, is not bound to its ACT domain, the catalytic site of PDT domain maintain open (active) state conformation as Sa-PDT structure. And the open state of its catalytic site become closed (allosterically inhibited) state if the regulatory molecule is bound to its ACT domain as Ct-PDT structure. However, the X-ray structure of prephenate dehydratase from Streptococcus mutans (Sm-PDT) shows that the catalytic site of Sm-PDT has closed state conformation without phenylalanine molecule bound to its regulatory site. The structure suggests a possibility that the binding of phenylalanine in its regulatory site may not be the only prerequisite for the closed state conformation of Sm-PDT.     

Purification of prephenate dehydratase from Corynebacterium glutamicum by affinity chromatography.

Prephenate dehydratase has been purified from the wild type strain Corynebacterium glutamicum by affinity chromatography. Three ligands, L-Trp, L-Tyr, and L-Phe have been tested as well as conditions for elution. L-Phe is the most specific ligand: it leads to a purification factor of 11 in one step using step gradients of NaCl in Tris-HCl buffer at pH 7.5.     

Regulation and state of aggregation of Bacillus subtilis prephenate dehydratase in the presence of allosteric effectors.

Prephenate dehydratase from Bacillus subtilis was found to exist in three states of aggregation. A high molecular weight (210,000) species was fully active and the catalytic activity was unaffected by the effectors methionine or phenylalanine. Low concentrations of phenylalanine caused dissociation to a Mr = 55,000 dimer. Heating to 32 degrees C also caused dissociation, but cooling and adding substrate or methionine favored association. When no effectors were present the enzyme eluted from Sephadex columns as a monomer. Both methionine and phenylalanine shifted the equilibrium from the inactive monomer to the active dimeric enzyme. In the presence of a saturating methionine concentration, the dimer possessed the same high activity as did the 210,000-dalton form. Phenylalanine inhibited the dimer, but not the higher molecular weight form. A model involving only three types of sites (catalytic, association-activation, and inhibition) is consistent with the data. It is proposed that phenylalanine is the preferred metabolite for binding both effector sites on the dimer; it binds the association-activation site with higher affinity than the inhibition site, but binding at the latter site has a greater effect on the catalytic rate. Methionine, like phenylalanine, has a hydrophobic side chain but is accommodated only at the association-activation site.     

Chorismate mutase/prephenate dehydrogenase from Escherichia coli K12: purification, characterization, and identification of a reactive cysteine

The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase/prephenate dehydrogenase, has been isolated from extracts of a regulatory mutant of Escherichia coli K12. The pure enzyme is a homodimer of total molecular weight 78 000 and displays Michaelis-Menten kinetics for both activities. Fingerprinting and amino acid sequencing of tryptic and thermolytic peptides of the S-[14C]carboxymethylated enzyme allowed the identification of three unique cysteine-containing sequences per subunit. Chemical modification of the native enzyme with 5,5'-dithiobis(2-nitrobenzoate) or iodoacetamide showed that one sulfhydryl group per subunit was particularly reactive, and the integrity of this group was essential for both enzymic activities. This work supports previous proposals for a close spatial relationship between the active sites.