Evolution of allosteric regulation in chorismate mutases from early plants.

Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 ŠX-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.

Design, selection, and characterization of a split chorismate mutase.

Split proteins are versatile tools for detecting protein-protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein-protein interactions.

Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum.

Chorismate mutase (CM) catalyses the rearrangement of chorismate to prephenate and is also the first and the key enzyme that diverges the shikimate pathway to either tryptophan (Trp) or phenylalanine (Phe) and tyrosine (Tyr). Corynebacterium glutamicum is one of the most important amino acid producers for the fermentation industry and has been widely investigated. However, the gene(s) encoding CM has not been experimentally identified in C. glutamicum. In this study, the ncgl0819 gene, which was annotated as 'conserved hypothetical protein' in the C. glutamicum genome, was genetically characterized to be essential for growth in minimal medium, and a mutant deleted of ncgl0819 was a Phe and Tyr auxotroph. Genetic cloning and expression of ncgl0819 in Escherichia coli resulted in the formation of a new protein (NCgl0819) having CM activity. It was concluded that ncgl0819 encoded the CM of C. glutamicum (CM0819). CM0819 was demonstrated to be a homodimer and is a new member of the monofunctional CMs of the AroQ structural class. The CM0819 activity was not affected by Phe, Tyr or Trp. Two 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthases (DS0950 and DS2098, formerly NCgl0950 and NCgl2098) had been previously identified from C. glutamicum. CM0819 significantly stimulated DAHP synthase (DS2098) activity. Physical interaction between CM0819 and DS2098 was observed. When CM0819 was present, DS2098 activity was subject to allosteric inhibition by chorismate and prephenate. Conserved hypothetical proteins homologous to CM0819 were identified in all known Corynebacterium genomes, suggesting a universal occurrence of CM0819-like CMs in the genus Corynebacterium.

Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved.

Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQ(gamma) subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5-15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000), Protein Sci. 9, 755-757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 A resolution. The crystals belong to space group P2(1), with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 angstroms, beta = 104.5 degrees. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.

Purified recombinant hypothetical protein coded by open reading frame Rv1885c of Mycobacterium tuberculosis exhibits a monofunctional AroQ class of periplasmic chorismate mutase activity.

Naturally occurring variants of the enzyme chorismate mutase are known to exist that exhibit diversity in enzyme structure, regulatory properties, and association with other proteins. Chorismate mutase was not annotated in the initial genome sequence of Mycobacterium tuberculosis (Mtb) because of low sequence similarity between known chorismate mutases. Recombinant protein coded by open reading frame Rv1885c of Mtb exhibited chorismate mutase activity in vitro. Biochemical and biophysical characterization of the recombinant protein suggests its resemblance to the AroQ class of chorismate mutases, prototype examples of which include the Escherichia coli and yeast chorismate mutases. We also demonstrate that unlike the corresponding proteins of E. coli, Mtb chorismate mutase does not have any associated prephenate dehydratase or dehydrogenase activity, indicating its monofunctional nature. The Rv1885c-encoded chorismate mutase showed allosteric regulation by pathway-specific as well as cross-pathway-specific ligands, as evident from proteolytic cleavage protection and enzyme assays. The predicted N-terminal signal sequence of Mtb chorismate mutase was capable of functioning as one in E. coli, suggesting that Mtb chorismate mutase belongs to the AroQ class of chorismate mutases. It was evident that Rv1885c may not be the only enzyme with chorismate mutase enzyme function within Mtb, based on our observation of the presence of chorismate mutase activity displayed by another hypothetical protein coded by open reading frame Rv0948c, a novel instance of the existence of two monofunctional chorismate mutases ever reported in any pathogenic bacterium.

The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa.

BACKGROUND: Chorismate mutases of the AroQ homology class are widespread in the Bacteria and the Archaea. Many of these exist as domains that are fused with other aromatic-pathway catalytic domains. Among the monofunctional AroQ proteins, that from Erwinia herbicola was previously shown to have a cleavable signal peptide and located in the periplasmic compartment. Whether or not this might be unique to E. herbicola was unknown. RESULTS: The gene coding for the AroQ protein was cloned from Salmonella typhimurium, and the AroQ protein purified from both S. typhimurium and Pseudomonas aeruginosa was shown to have a periplasmic location. The periplasmic chorismate mutases (denoted *AroQ) are shown to be a distinct subclass of AroQ, being about twice the size of cytoplasmic AroQ proteins. The increased size is due to a carboxy-terminal extension of unknown function. In addition, a so-far novel aromatic aminotransferase was shown to be present in the periplasm of P. aeruginosa. CONCLUSIONS: Our analysis has detected a number of additional *aroQ genes. The joint presence of *AroQ, cyclohexadienyl dehydratase and aromatic aminotransferase in the periplasmic compartment of P. aeruginosa comprises a complete chorismate-to-phenylalanine pathway and accounts for the "hidden overflow pathway" to phenylalanine described previously.

Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain.

The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe. To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E. coli strain NK6024, and studied for their effect on PDT activity. Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy. Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry. In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L. PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe. Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis. The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein. The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein. These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism. To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala. None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis. However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding. For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower.

Chorismate mutase and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the methylotrophic actinomycete Amycolatopsis methanolica.

Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII and CMII synthesis to L-Phe repression was isolated. DSII was partially purified (a 42-kDa protein); its activity was strongly inhibited by L-Tyr. CMII was purified to homogeneity (93.6 fold) and characterized as a homodimeric protein with 16-kDa subunits, completely insensitive to feedback inhibition by L-Phe and L-Tyr. The activity of CMII was activated by CMIb; the activity of CMII plus CMIb was again inhibited by L-Phe and L-Tyr. A tightly blocked L-Phe- plus L-Tyr-requiring derivative of mutant strain GH141, GH141-19, that had lost both CMIa and CMII activities was isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structures of the monofunctional chorismate mutase from Bacillus subtilis and its complex with a transition state analog.

We have solved the structure of a chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5), the 1.9-A crystal structure of the monofunctional enzyme from Bacillus subtilis. The structure determination process was an unusual one, involving 12 monomers of the enzyme in the asymmetric unit. This structure was solved by the multiple isomorphous replacement method with partial structure phase combination and molecular averaging. The final model, which includes 1380 residues and 522 water molecules in an asymmetric unit, has been refined at 1.9 A and the current crystallographic R value is 0.201. The B. subtilis chorismate mutase is a homotrimer, with beta-sheets from each monomer packing to form the core of a pseudo-alpha beta-barrel with helices on the outside of the trimer. In addition, the active sites have been located by using data from a complex with an endo-oxabicyclic inhibitor that mimics the transition state of the reaction. The structure of this complex has been refined to 2.2 A with a current R value of 0.182 for a model that includes 1388 residues, 12 inhibitor molecules, and 530 water molecules in the asymmetric unit. In each trimer, three equivalent active sites are located at the interfaces of two adjacent subunits.

The aroQ-encoded monofunctional chorismate mutase (CM-F) protein is a periplasmic enzyme in Erwinia herbicola.

Enteric bacteria possess two species of chorismate mutase which exist as catalytic domains on the amino termini of the bifunctional PheA and TyrA proteins. In addition, some of these organisms possess a third chorismate mutase, CM-F, which exists as a small monofunctional protein. The CM-F gene (denoted aroQ) from Erwinia herbicola was cloned and sequenced for the first time. A strategy for selection by functional complementation in a chorismate mutase-free Escherichia coli background was devised by using a recombinant plasmid derivative of pUC18 carrying a Zymomonas mobilis tyrC insert which encodes cyclohexadienyl dehydrogenase. The aroQ gene is 543 bp in length, predicting a 181-residue protein product having a calculated molecular mass of 20,299 Da. The E. herbicola aroQ promoter is recognized by E. coli, and a putative sigma-70 promoter region was identified. N-terminal amino acid sequencing of the purified CM-F protein indicated cleavage of a 20-residue signal peptide. This was consistent with the monomeric molecular mass determined for the enzyme of about 18,000 Da. The native enzyme is a homodimer. The implied translocation of CM-F was confirmed by osmotic shock experiments which demonstrated a periplasmic location. Immunogold electron microscopy indicated a polar localization within the periplasm. Polyclonal antibody raised against E. herbicola CM-F did not cross-react with the CM-F protein from the closely related Serratia rubidaea, as well as from a number of other gram-negative bacteria. Furthermore, when the E. herbicola aroQ gene was used as a probe in Southern blot hybridizations with EcroRI digests of chromosomal DNA from S. rubidaea and other enteric organisms, no hybridization was detected at low stringency. Thus, the aroQ gene appears to be unusually divergent among closely related organisms. The deduced CM-F amino acid sequence did not exhibit compelling evidence for homology with the monofunctional chorismate mutase protein of Bacillus subtilis.

13C NMR studies of the enzyme-product complex of Bacillus subtilis chorismate mutase.

The chorismate mutase reaction is a rare enzyme-catalyzed 3,3-sigmatropic rearrangement of chorismate to prephenate. Bacillus subtilis chorismate mutase was overproduced and purified from Escherichia coli XL1-Blue (pBSCM2) using a modification of the procedure of Gray et al. (Gray, J. V., Grolinelli-Pimpaneau, B., & Knowles, J. R. (1990) Biochemistry 29, 376-383); the modification leads to minimal contaminating prephenate dehydratase activity (< 0.001%). The native molecular mass of B. subtilis chorismate mutase was determined by gel filtration to be approximately 44 kDa, indicative of a homotrimer of the 14.5-kDa subunits as determined by electrospray mass spectrometry. 13C NMR was used to study the structure of [U-13C]prephenate bound at the active site of B. subtilis chorismate mutase. All the enzyme-bound 13C NMR resonances of [U-13C]prephenate were assigned, and where possible, 1JC,Cs were quantified; [1,3,5,8-13C]prephenate and [2,6,9-13C]prephenate, prepared respectively from [1,3,5,8-13C]chorismate and [2,6,9-13C]chorismate, aided the 13C NMR resonance assignments. Enzyme-bound prephenate exhibits remarkably different chemical shifts relative to free prephenate; the chemical shift changes range from -6.6 ppm for the C6 resonance to 5.6 ppm for the C5 resonance, suggesting a strong perturbation of the C5-C6 bond. 13C NMR studies of model compounds at various pH values and in various solvents suggest that the observed 13C chemical shift changes of enzyme-bound prephenate cannot be rationalized solely on the basis of changes in the pKas of the carboxylic acid groups or hydrophobic solvation at the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Monofunctional chorismate mutase from Serratia rubidaea: a paradigm system for the three-isozyme gene family of enteric bacteria.

Serratia rubidaea (ATCC 27614) typifies a substantial number of enteric bacteria which, unlike Escherichia coli, possess a monofunctional species of chorismate mutase (denoted CM-F). CM-F coexists with two additional species of chorismate mutase, each of the latter being one catalytic domain of a bifunctional protein. The two bifunctional proteins are utilized for phenylalanine (CM-P/prephenate dehydratase) and tyrosine (CM-T/cyclohexadienyl dehydrogenase) biosynthesis in all enteric bacteria. S. rubidaea was selected as the organism of choice for purification of CM-F because of the relatively abundant level of expression found for this enzyme. The monofunctional CM-F enzyme was purified about 1600-fold with a yield of about 16%. This is the first monofunctional chorismate mutase to be purified from any gram-negative prokaryote. The CM-F enzyme is a positively charged homodimer made up of 20-kDa subunits. It has a pH optimum of 5.5, exhibits a Km value of 0.33 mM for chorismate, and is sensitive to product inhibition by prephenate that is competitive with respect to chorismate. It is insensitive to feedback inhibition by any of the aromatic amino acids. Partial purification of the bifunctional P-protein and the bifunctional T-protein was also carried out in order to compare the properties of CM-F, CM-P, and CM-T in a common organism. The most striking differential properties of the three isozymes were those of pH optimum and degree of protection conferred by dithiothreitol.

Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution.

Chorismate mutase, a branch-point enzyme in the aromatic amino acid pathway of Saccharomyces cerevisiae, and also a mutant chorismate mutase with a single amino acid substitution in the C-terminal part of the protein have been purified approximately 20-fold and 64-fold from overproducing strains, respectively. The wild-type enzyme is activated by tryptophan and subject to feedback inhibition by tyrosine, whereas the mutant enzyme does not respond to activation by tryptophan nor inhibition by tyrosine. Both enzymes are dimers consisting of two identical subunits of Mr 30,000, each one capable of binding one substrate and one activator molecule. Each subunit of the wild-type enzyme also binds one inhibitor molecule, whereas the mutant enzyme lost this ability. The enzyme reaction was observed by 1H NMR and shows a direct and irreversible conversion of chorismate to prephenate without the accumulation of any enzyme-free intermediates. The kinetic data of the wild-type chorismate mutase show positive cooperativity toward the substrate with a Hill coefficient of 1.71 and a [S]0.5 value of 4.0 mM. In the presence of the activator tryptophan, the cooperativity is lost. The enzyme has an [S]0.5 value of 1.2 mM in the presence of 10 microM tryptophan and an increased [S]0.5 value of 8.6 mM in the presence of 300 microM tyrosine. In the mutant enzyme, a loss of cooperativity was observed, and [S]0.5 was reduced to 1.0 mM. This enzyme is therefore locked in the activated state by a single amino acid substitution.

Monofunctional chorismate mutase from Bacillus subtilis: purification of the protein, molecular cloning of the gene, and overexpression of the gene product in Escherichia coli.

The monofunctional chorismate mutase from Bacillus subtilis has been purified 2200-fold to homogeneity. The enzyme is a homodimer of subunit Mr = 14,500 and is the smallest natural chorismate mutase that has been characterized. The purified enzyme follows Michaelis-Menten kinetics with a Km of 100 microM and a kcat of 50 s-1, carries no other associated enzymic activities, and is unaffected by any of the aromatic amino acids. The N-terminal amino acid sequence of the protein has been determined, and this information has been used to construct a precise oligonucleotide probe for the gene by means of in vitro DNA amplification from total chromosomal DNA by the polymerase chain reaction. The cloned aroH gene encodes a protein of 127 amino acid residues and is expressed in Escherichia coli. The cloned gene product is indistinguishable from that purified from Bacillus. The aroH coding region was directly subcloned into a phagemid expression vector by means of the polymerase chain reaction. The resulting construct, with the aroH gene positioned behind efficient transcription and translation initiation sequences of E. coli, results in the production of the monofunctional mutase at levels of 30-35% of the soluble cell protein in E. coli transformants. Chorismate mutases comprise a set of functionally related proteins that show little sequence similarity to each other. This diversity stands in contrast to other chorismate-utilizing enzymes.

Terminal phenylalanine and tyrosine biosynthesis of Microtetraspora glauca.

The enzymes of the terminal steps of the phenylalanine and tyrosine biosynthesis were partially purified and characterized in Microtetraspora glauca, a spore-forming member of the order Actinomycetales. This bacterium relies exclusively on the phenylpyruvate route for phenylalanine synthesis, no arogenate dehydratase activity being found. Prephenate dehydratase is subject to feedback inhibition by phenylalanine, tyrosine and tryptophan, each acting as competitive inhibitor by increasing the Km of 72 microM for prephenate. Based on the results of gel chromatography on Sephadex G-200, the molecular mass of about 110,000 Da is not altered by any of the effectors. The enzyme is quite sensitive to inhibition by 4-hydroxymercuribenzoate. Microtetraspora glauca can utilize arogenate and 4-hydroxyphenylpyruvate as intermediates in tyrosine biosynthesis. Prephenate and arogenate dehydrogenase activities copurifying from ion exchange columns with coincident profiles were detected. From gel-filtration columns the two activities eluted at an identical molecular-mass position of about 68,000 Da. The existence of a single protein exhibiting substrate ambiguity is consistent with the findings, that both dehydrogenases have similar chromatographic properties, exhibit cofactor requirement for NAD and are inhibited to the same extent by tyrosine and 4-hydroxymercuribenzoate.

The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa.

The enzyme chorismate synthase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroC gene and confirmed by determining the N-terminal amino acid sequence of the purified enzyme. The complete polypeptide chain consists of 357 amino acid residues and has a calculated subunit Mr of 38,183. Cross-linking and gel-filtration experiments show that the enzyme is tetrameric. An improved purification of chorismate synthase from Neurospora crassa is also described. Cross-linking and gel-filtration experiments on the N. crassa enzyme show that it is also tetrameric with a subunit Mr of 50,000. It is proposed that the subunits of the N. crassa enzyme are larger because they contain a diaphorase domain that is absent from the E. coli enzyme.

Purification and characterization of an inducible aromatic amino acid-sensitive form of chorismate mutase from Solanum tuberosum L. tubers.

An amino acid-sensitive form of chorismate mutase (CM) has been purified over 1000-fold from disks excised from tubers of Solanum tuberosum L. cv White Rose. Purification was accomplished by chromatography on Matrix Blue A followed by affinity chromatography with tryptophan as ligand. CM assays performed in the absence of tryptophan yielded pH-dependent sigmoidal kinetics. At pH 8.0, sigmoidal kinetics were observed with a Hill coefficient of 1.66 (S0.5 = 188 microM). However, a shift from sigmoidal to hyperbolic kinetics was observed when assays were performed at pH 8.5. Addition of 9 microM tryptophan to the assay resulted in maximum activation of the enzyme with a Ka of 1.2 microM. When assayed in the presence of tryptophan, hyperbolic kinetics were observed over the pH range 6.0-8.0. Addition of tryptophan also decreased the Km for chorismate from 185 to 45 microM. Tryptophan (0.1 mM) completely protected CM from inhibition by phenylalanine (1.8 mM) and tyrosine (1.8 mM). However, in the absence of the activator, phenylalanine and tyrosine exhibited 50% inhibition at 0.80 and 0.68 mM concentrations, respectively. Both phenylalanine and tyrosine competitively inhibited CM activity with Ki values of 550 and 440 mM, respectively. Arogenate (1.0 mM) had no effect on CM activity in either the presence or absence of tryptophan. Analytical isoelectric focusing yielded an isoelectric point of 4.73.

Chorismate mutase isoenzymes from Sorghum bicolor: purification and properties.

Two forms of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, have been detected in etiolated seedlings of Sorghum bicolor after DEAE-cellulose chromatography. CM-1 and CM-2 contained 44 and 56%, respectively, of the total activity measured after DEAE-cellulose chromatography. CM-1 was activated by tryptophan and inhibited by phenylalanine and tyrosine. In contrast, CM-2 was insensitive to all three aromatic amino acids. CM-1 and CM-2 were purified 1389- and 1018-fold, respectively, by anion exchange, hydrophobic, and dye matrix chromatography. The molecular weights estimated by gel filtration on Sephacryl S-200 were 56,000 for CM-1 and 48,000 for CM-2. Subunit molecular weights of the two forms were estimated by sodium dodecyl sulfate-gel electrophoresis at 36,000 and 51,000 for CM-1 and CM-2, respectively. Tryptophan was required for the stability of CM-1 at all stages of purification. Both isoenzymes were stable at 0 or -20 degrees C and had broad pH optima (6-10 for CM-1 and 7.5-9.5 for CM-2).

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.