NORMAL GENOMIC

• Prakash P, Pathak N, Hasnain SE
• pheA (Rv3838c) of Mycobacterium tuberculosis encodes an allostericallyregulated monofunctional prephenate dehydratase that requires bothcatalytic and regulatory domains for optimum activity.
• J Biol Chem. 2005; 280: 20666-71
• Display abstract

• Prephenate dehydratase (PDT) is a key regulatory enzyme in l-phenylalaninebiosynthesis. In Mycobacterium tuberculosis, expression of pheA, the geneencoding PDT, has been earlier reported to be iron-dependent (1, 2). Wereport that M. tuberculosis pheA is also regulated at the protein level byaromatic amino acids. All of the three aromatic amino acids(phenylalanine, tyrosine, and tryptophan) are potent allosteric activatorsof M. tuberculosis PDT. We also provide in vitro evidence that M.tuberculosis PDT does not possess any chorismate mutase activity, whichsuggests that, unlike many other enteric bacteria (where PDT exists as afusion protein with chorismate mutase), M. tuberculosis PDT is amonofunctional and a non-fusion protein. Finally, the biochemical andbiophysical properties of the catalytic and regulatory domains (ACTdomain) of M. tuberculosis PDT were studied to observe that, in theabsence of the ACT domain, the enzyme not only loses its regulatoryactivity but also its catalytic activity. These novel results provideevidence for a monofunctional prephenate dehydratase enzyme from apathogenic bacterium that exhibits extensive allosteric activation byaromatic amino acids and is absolutely dependent upon the presence ofcatalytic as well as the regulatory domains for optimum enzyme activity.

• Chen S, Vincent S, Wilson DB, Ganem B
• Mapping of chorismate mutase and prephenate dehydrogenase domains in theEscherichia coli T-protein.
• Eur J Biochem. 2003; 270: 757-63
• Display abstract

• The Escherichia coli bifunctional T-protein transforms chorismic acid top-hydroxyphenylpyruvic acid in the l-tyrosine biosynthetic pathway. The373 amino acid T-protein is a homodimer that exhibits chorismate mutase(CM) and prephenate dehydrogenase (PDH) activities, both of which arefeedback-inhibited by tyrosine. Fifteen genes coding for the T-protein andvarious fragments thereof were constructed and successfully expressed inorder to characterize the CM, PDH and regulatory domains. Residues 1-88constituted a functional CM domain, which was also dimeric. Both the PDHand the feedback-inhibition activities were localized in residues 94-373,but could not be separated into discrete domains. The activities of clonedCM and PDH domains were comparatively low, suggesting some cooperativeinteractions in the native state. Activity data further indicate that thePDH domain, in which NAD, prephenate and tyrosine binding sites werepresent, was more unstable than the CM domain.

• Zhang S, Wilson DB, Ganem B
• An engineered chorismate mutase with allosteric regulation.
• Bioorg Med Chem. 2003; 11: 3109-14
• Display abstract

• Besides playing a central role in phenylalanine biosynthesis, thebifunctional P-protein in Eschericia coli provides a unique model systemfor investigating whether allosteric effects can be engineered intoprotein catalysts using modular regulatory elements. Previous studies haveestablished that the P-protein contains three distinct domains whosefunctions are preserved, even when separated: chorismate mutase (residues1-109), prephenate dehydratase (residues 101-285), and an allostericdomain (residues 286-386) for feedback inhibition by phenylalanine. Bydeleting the prephenate dehydrase domain, a functional chorismate mutaselinked directly to the phenylalanine binding domain has been engineeredand overexpressed. This manuscript reports the catalytic properties of themutase in the absence and presence of phenylalanine.

• Kloosterman H, Hessels GI, Vrijbloed JW, Euverink GJ, Dijkhuizen L
• (De)regulation of key enzyme steps in the shikimate pathway andphenylalanine-specific pathway of the actinomycete Amycolatopsismethanolica.
• Microbiology. 2003; 149: 3321-30
• Display abstract

• Prephenate dehydratase (PDT), chorismate mutase (CM) and3-deoxy-D-arabino-7-heptulosonate 7-phosphate (DAHP) synthase are keyregulatory enzymes in aromatic amino acid biosynthesis in the actinomyceteAmycolatopsis methanolica. Deregulated, feedback-control-resistant mutantswere isolated by incubation of A. methanolica on glucose mineral agarcontaining the toxic analogue p-fluoro-DL-phenylalanine (pFPhe). Severalof these mutants had completely lost PDT sensitivity to Phe inhibition andTyr activation. Mutant characterization yielded new information about PDTamino acid residues involved in Phe and Tyr effector binding sites. A.methanolica wild-type cells grown on glucose mineral medium normallypossess a bifunctional CM/DAHP synthase protein complex (with DS1, aplant-type DAHP synthase). The CM activity of this protein complex isfeedback-inhibited by Tyr and Phe, while DS1 activity is mainly inhibitedby Trp. Isolation of pFPhe-resistant mutants yielded twofeedback-inhibition-resistant CM mutants. These were characterized asregulatory mutants, derepressed in (a) synthesis of CM, now occurring asan abundant, feedback-inhibition-resistant, separate protein, and (b)synthesis of an alternative DAHP synthase (DS2, an E. coli-type DAHPsynthase), only inhibited by Tyr and Trp. DS1 and DS2 thus are wellintegrated in A. methanolica primary metabolism: DS1 and CM form a proteincomplex, which stimulates CM activity and renders it sensitive to feedbackinhibition by Phe and Tyr. Synthesis of CM and DS2 proteins appears to becontrolled co-ordinately, sensitive to Phe-mediated feedback repression.

• Helmstaedt K, Heinrich G, Lipscomb WN, Braus GH
• Refined molecular hinge between allosteric and catalytic domain determinesallosteric regulation and stability of fungal chorismate mutase.
• Proc Natl Acad Sci U S A. 2002; 99: 6631-6
• Display abstract

• The yeast chorismate mutase is regulated by tyrosine as feedback inhibitorand tryptophan as crosspathway activator. The monomer consists of acatalytic and a regulatory domain covalently linked by the loop L220s(212-226), which functions as a molecular hinge. Two monomers form theactive dimeric enzyme stabilized by hydrophobic interactions in thevicinity of loop L220s. The role of loop L220s and its environment forenzyme regulation, dimerization, and stability was analyzed. Substitutionof yeast loop L220s in place of the homologous loop from the correspondingand similarly regulated Aspergillus enzyme (and the reverse substitution)changed tyrosine inhibition to activation. Yeast loop L220s substitutedinto the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme.Monomeric yeast chorismate mutases could be generated by substituting twohydrophobic residues in and near the hinge region. The resultingThr-212-->Asp-Phe-28-->Asp enzyme was as stable as wild type, but lostallosteric regulation and showed reduced catalytic activity. These resultsunderline the crucial role of this molecular hinge for inhibition,activation, quaternary structure, and stability of yeast chorismatemutase.

• Zhang S, Wilson DB, Ganem B
• Probing the catalytic mechanism of prephenate dehydratase by site-directedmutagenesis of the Escherichia coli P-protein dehydratase domain.
• Biochemistry. 2000; 39: 4722-8
• Display abstract

• The Escherichia coli bifunctional P-protein, which plays a central role inL-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 mechanismof PDT in the P-protein, 24 mutations of 15 conserved residues in the PDTdomain were created, expressed in the pheA(-)E. coli strain NK6024, andstudied for their effect on PDT activity. Fourteen mutant enzymes werepurified to homogeneity, tested for feedback inhibition by Phe, andcharacterized by kinetic analysis and circular dichroism spectroscopy.Selected mutant enzymes were further studied by gel filtration,fluorescence emission, and microcalorimetry. In addition, a monofunctionalPDT domain (PDT20, residues 101-285) was cloned and overexpressed inplasmid pET with expression levels up to 200-250 mg/L. PDT20 retained fullPDT activity, lacked CM activity, and was insensitive to feedbackinhibition by Phe. Four residues (T278, N160, Q215, and S208) were shownto be important for PDT catalysis. The values of k(cat)/K(m) for theS208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lowerfor the N160A and Q215A mutant enzymes than the wild-type (WT) protein.The T278A and T278V mutant enzymes displayed no measurable catalyticactivity, yet bound both prephenate and a competitive inhibitor (S-DNBA)comparably to the WT protein. These data, taken together with the normalCD spectra of the mutant enzymes, strongly suggested that T278 wasinvolved in the catalytic mechanism. To establish whether acidic residueswere involved in catalysis, all the conserved Glu and Asp residues in thePDT domain were mutated to Ala. None of these mutations significantlyreduced PDT activity, indicating that the acidic residues of the PDTdomain are not directly involved in catalysis. However, two mutant enzymes(E159A and E232A) displayed higher levels of PDT activity (2.2- and3.5-fold, respectively), which was due to enhanced substrate binding. Forthe double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca. 7-fold higherthan for the WT enzyme, while its K(m) was 4.6-fold lower.

• Pohnert G, Zhang S, Husain A, Wilson DB, Ganem B
• Regulation of phenylalanine biosynthesis. Studies on the mechanism ofphenylalanine binding and feedback inhibition in the Escherichia coliP-protein.
• Biochemistry. 1999; 38: 12212-7
• Display abstract

• Isothermal titration calorimetry (ITC) and site-directed mutagenesis wereused to study the interaction of Phe with (a) the Escherichia coliP-protein, a bifunctional chorismate mutase/prephenate dehydratase that isfeedback inhibited by Phe, (b) PDT32, a 32 kDa P-protein fragment(residues 101-386) containing the prephenate dehydratase and regulatorydomains, and (c) R12, a C-terminal 12 kDa P-protein fragment (residues286-386) containing the regulatory domain. DeltaH(total) values for PDT32,which included the heats of Phe binding, conformational change, anddimerization, established that in developing a mechanism for end productfeedback inhibition, the P-protein has evolved a ligand recognition domainthat exhibits Phe-binding enthalpies comparable to those reported forother full-fledged amino acid receptor proteins. Sequence alignments ofR12 with other Phe-binding enzymes identified two highly conservedregions, GALV (residues 309-312) and ESRP (residues 329-332).Site-directed mutagenesis and ITC established that changes in the GALV andESRP regions affected Phe binding and feedback inhibition to differentextents. Mutagenesis further showed that C374 was essential for feedbackinhibition, but not for Phe binding, while W338 was involved in Phebinding, but not in the Phe-induced conformational change required forfeedback inhibition.

• Schnappauf G, Lipscomb WN, Braus GH
• Separation of inhibition and activation of the allosteric yeast chorismatemutase.
• Proc Natl Acad Sci U S A. 1998; 95: 2868-73
• Display abstract

• Yeast chorismate mutase (EC 5.4.99.5) shows homotropic activation by thesubstrate, allosteric activation by tryptophan, and allosteric inhibitionby tyrosine. In this study mutants of chorismate mutase have been foundthat remain sensitive to one allosteric effector (tryptophan) butinsensitive to the other (tyrosine). These mutations are located in thecatalytic domain: loop 220s (212-226) and helix 12 (227-251). The firstexample starts with the Thr-266 --> Ile mutant that had previously beenshown to be locked in the activated R state. The additional mutationIle-225 --> Thr unlocks the R state and restores the activation bytryptophan but not the inhibition by tyrosine. The second example refersto a molecular trigger for the switch between the T and R state: ahydrogen-bonded system, which stabilizes only the T state, from Tyr-234 toGlu-23 to Arg-157. Various mutants of Tyr-234, especially Tyr-234 --> Phe,are unresponsive to tyrosine but are activated by tryptophan. Thisseparation of activation from inhibition may indicate a pathway foractivation that is independent of the allosteric transition and may alsobe consistent with an intermediate structure between T and R states.

• Strater N, Schnappauf G, Braus G, Lipscomb WN
• Mechanisms of catalysis and allosteric regulation of yeast chorismatemutase from crystal structures.
• Structure. 1997; 5: 1437-52
• Display abstract

• BACKGROUND: Chorismate mutase (CM) catalyzes the Claisen rearrangement ofchorismate to prephenate, notably the only known enzymatically catalyzedpericyclic reaction in primary metabolism. Structures of the enzyme incomplex with an endo-oxabicyclic transition state analogue inhibitor,previously determined for Bacillus subtilis and Escherichia coli CM,provide structural insight into the enzyme mechanism. In contrast to thesebacterial CMs, yeast CM is allosterically regulated in two ways:activation by tryptophan and inhibition by tyrosine. Yeast CM exists intwo allosteric states, R (active) and t (inactive). RESULTS: We havedetermined crystal structures of wild-type yeast CM cocrystallized withtryptophan and an endo-oxabicyclic transition state analogue inhibitor, ofwild-type yeast CM co-crystallized with tyrosine and the endo-oxabicyclictransition state analogue inhibitor and of the Thr226-->Ser mutant ofyeast CM in complex with tryptophan. Binding of the transition stateanalogue inhibitor to CM keeps the enzyme in a 'super R' state, even ifthe inhibitory effector tyrosine is bound to the regulatory site.CONCLUSIONS: The endo-oxabicyclic inhibitor binds to yeast CM in a similarway as it does to the distantly related CM from E. coli. Theinhibitor-binding mode supports a mechanism by which polar sidechains ofthe enzyme bind the substrate in the pseudo-diaxial conformation, which isrequired for catalytic turnover. A lysine and a protonated glutamatesidechain have a critical role in the stabilization of the transitionstate of the pericyclic reaction. The allosteric transition from T-->Rstate is accompanied by a 15 degrees rotation of one of the two subunitsrelative to the other (where 0 degrees rotation defines the T state). Thisrotation causes conformational changes at the dimer interface which aretransmitted to the active site. An allosteric pathway is proposed toinclude residues Phe28, Asp24 and Glu23, which move toward the activesitecavity in the T state. In the presence of the transition-state analogue asuper R state is formed, which is characterised by a 22 degrees rotationof one subunit relative to the other.

• Schnappauf G, Strater N, Lipscomb WN, Braus GH
• A glutamate residue in the catalytic center of the yeast chorismate mutaserestricts enzyme activity to acidic conditions.
• Proc Natl Acad Sci U S A. 1997; 94: 8491-6
• Display abstract

• Chorismate mutase acts at the first branchpoint of aromatic amino acidbiosynthesis and catalyzes the conversion of chorismate to prephenate.Comparison of the x-ray structures of allosteric chorismate mutase fromthe yeast Saccharomyces cerevisiae with Escherichia coli chorismatemutase/prephenate dehydratase suggested conserved active sites betweenboth enzymes. We have replaced all critical amino acid residues, Arg-16,Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismatemutase by aliphatic amino acid residues. The resulting enzymes exhibit thenecessity of these residues for catalytic function and provide evidence oftheir localization at the active site. Unlike some bacterial enzymes,yeast chorismate mutase has highest activity at acidic pH values.Replacement of Glu-246 in the yeast chorismate mutase by glutamine changesthe pH optimum for activity of the enzyme from a narrow to a broad pHrange. These data suggest that Glu-246 in the catalytic center must beprotonated for maximum catalysis and restricts optimal activity of theenzyme to low pH.

• Strater N, Hakansson K, Schnappauf G, Braus G, Lipscomb WN
• Crystal structure of the T state of allosteric yeast chorismate mutase andcomparison with the R state.
• Proc Natl Acad Sci U S A. 1996; 93: 3330-4
• Display abstract

• The crystal structure of the tyrosine-bound T state of allosteric yeastSaccharomyces cerevisiae chorismate mutase was solved by molecularreplacement at a resolution of 2.8 angstroms using a monomer of theR-state structure as the search model. The allosteric inhibitor tyrosinewas found to bind in the T state at the same binding site as theallosteric activator tryptophan binds in the R state, thus defining oneregulatory binding site for each monomer. Activation by tryptophan iscaused by the larger steric size of its side chain, thereby pushing apartthe allosteric domain of one monomer and helix H8 of the catalytic domainof the other monomer. Inhibition is caused by polar contacts of tyrosinewith Arg-75 and Arg-76 of one monomer and with Gly-141, Ser-142, andThr-145 of the other monomer, thereby bringing the allosteric andcatalytic domains closer together. The allosteric transition includes an 8degree rotation of each of the two catalytic domains relative to theallosteric domains of each monomer (domain closure). Alternatively, thistransition can be described as a 15 degree rotation of the catalyticdomains of the dimer relative to each other.

• Graf R, Dubaquie Y, Braus GH
• Modulation of the allosteric equilibrium of yeast chorismate mutase byvariation of a single amino acid residue.
• J Bacteriol. 1995; 177: 1645-8
• Display abstract

• Chorismate mutase (EC 5.4.99.5) from the yeast Saccharomyces cerevisiae isan allosteric enzyme which can be locked in its active R (relaxed) stateby a single threonine-to-isoleucine exchange at position 226. Seven newreplacements of residue 226 reveal that this position is able to directthe enzyme's allosteric equilibrium, without interfering with thecatalytic constant or the affinity for the activator.

• Xue Y, Lipscomb WN
• Location of the active site of allosteric chorismate mutase fromSaccharomyces cerevisiae, and comments on the catalytic and regulatorymechanisms.
• Proc Natl Acad Sci U S A. 1995; 92: 10595-8
• Display abstract

• The active site of the allosteric chorismate mutase (chorismatepyruvatemutase, EC 5.4.99.5) from yeast Saccharomyces cerevisiae (YCM) waslocated by comparison with the mutase domain (ECM) of chorismatemutase/prephenate dehydratase [prephenate hydro-lyase (decarboxylating),EC 4.2.1.51] (the P protein) from Escherichia coli. Active site domains ofthese two enzymes show very similar four-helix bundles, each of 94residues which superimpose with a rms deviation of 1.06 A. Of the sevenactive site residues, four are conserved: the two arginines, which bind tothe inhibitor's two carboxylates; the lysine, which binds to the etheroxygen; and the glutamate, which binds to the inhibitor's hydroxyl groupin ECM and presumably in YCM. The other three residues in YCM (ECM) areThr-242 (Ser-84), Asn-194 (Asp-48), and Glu-246 (Gln-88). This Glu-246,modeled close to the ether oxygen of chorismate in YCM, may function as apolarizing or ionizable group, which provides another facet to thecatalytic mechanism.

• Xue Y, Lipscomb WN, Graf R, Schnappauf G, Braus G
• The crystal structure of allosteric chorismate mutase at 2.2-A resolution.
• Proc Natl Acad Sci U S A. 1994; 91: 10814-8
• Display abstract

• The crystal structure of an allosteric chorismate mutase, theThr-226-->Ile mutant, from yeast Saccharomyces cerevisiae has beendetermined to 2.2-A resolution by using the multiple isomorphousreplacement method. Solvent-flattening and electron-density modificationwere applied for phase improvement. The current crystallographic R factoris 0.196. The final model includes 504 of the 512 residues and 97 watermolecules. In addition, two tryptophan molecules were identified in theinterface between monomers. The overall structure is completely differentfrom the reported structure of chorismate mutase from Bacillus subtilis.This structure showed 71% helices with essentially no beta-sheetstructures.

• Ramilo C, Braus G, Evans JN
• A tyrosine residue is involved in the allosteric binding of tryptophan toyeast chorismate mutase.
• Biochim Biophys Acta. 1993; 1203: 71-6
• Display abstract

• Comparative 1H-NMR studies have been carried out on wild-type chorismatemutase, which is activated by tryptophan and inhibited by tyrosine andphenylalanine, and mutant yeast chorismate mutase, which has a singlepoint mutation (T226I) and is not allosterically regulated but 'locked' inthe activated state. Double quantum-filtered COSY spectra show cross-peakswhich have been assigned to a tyrosine that are absent in the mutantenzyme and in the wild-type enzyme plus tryptophan when compared with thewild-type enzyme alone. These observations indicate the involvement of atyrosine at or near the allosteric binding site. The involvement oftyrosine in tryptophan binding was tested by modification of tyrosine inyeast chorismate mutase by nitration with tetranitromethane. All forms ofthe enzyme exhibited an approx. 50% reduction in specific activity, but itwas found that preincubation of the wild-type with the allostericactivator, tryptophan, lead to partial protection against loss in specificactivity. Only one tyrosine residue was nitrated in the wild-type enzymeand this tyrosine was identified by tryptic digestion and sequencing, andfound to be very close to the site of the single point mutation in themutant enzyme. It is proposed that Tyr-234 is located at or near theallosteric activation site.

• Xia T, Zhao G, Jensen RA
• Loss of allosteric control but retention of the bifunctional catalyticcompetence of a fusion protein formed by excision of 260 base pairs fromthe 3' terminus of pheA from Erwinia herbicola.
• Appl Environ Microbiol. 1992; 58: 2792-8
• Display abstract

• A bifunctional protein denoted as the P protein and encoded by pheA iswidely present in purple gram-negative bacteria. This P protein carriescatalytic domains that specify chorismate mutase (CM-P) and prephenatedehydratase. The instability of a recombinant plasmid carrying a pheAinsert cloned from Erwinia herbicola resulted in a loss of 260 bp plus theTAA stop codon from the 3' terminus of pheA. The plasmid carrying thetruncated pheA gene (denoted pheA*) was able to complement an Escherichiacoli pheA auxotroph. pheA* was shown to be a chimera composed of theresidual 5' part of pheA (901 bp) and a 5-bp fragment from the pUC18vector. The new fusion protein (PheA*) retained both chorismate mutase andprephenate dehydratase activities. PheA* had a calculated subunitmolecular weight of 33,574, in comparison to the 43,182-molecular-weightsubunit size of PheA. The deletion did not affect the ability of PheA* toassume the native dimeric configuration of PheA. Both the CM-P andprephenate dehydratase components of PheA* were insensitive toL-phenylalanine inhibition, in contrast to the corresponding components ofPheA. L-Phenylalanine protected both catalytic activities of PheA fromthermal inactivation, and this protective effect of L-phenylalanine uponthe PheA* activities was lost. PheA* was more stable than PheA to thermalinactivation; this was more pronounced for prephenate dehydratase than forCM-P. In the presence of dithiothreitol, the differential resistance ofPheA* prephenate dehydratase to thermal inactivation was particularlystriking.(ABSTRACT TRUNCATED AT 250 WORDS)

• Fischer RS, Zhao G, Jensen RA
• Cloning, sequencing, and expression of the P-protein gene (pheA) ofPseudomonas stutzeri in Escherichia coli: implications for evolutionaryrelationships in phenylalanine biosynthesis.
• J Gen Microbiol. 1991; 137: 1293-301
• Display abstract

• The pheA gene encoding the bifunctional P-protein (chorismatemutase:prephenate dehydratase) was cloned from Pseudomonas stutzeri andsequenced. This is the first gene of phenylalanine biosynthesis to becloned and sequenced from Pseudomonas. The pheA gene was expressed inEscherichia coli, allowing complementation of an E. coli pheA auxotroph.The enzymic and physical properties of the P-protein from a recombinant E.coli auxotroph expressing the pheA gene were identical to those of thenative enzyme from P. stutzeri. The nucleotide sequence of the P. stutzeripheA gene was 1095 base pairs in length, predicting a 365-residue proteinproduct with an Mr of 40,844. Codon usage in the P. stutzeri pheA gene wassimilar to that of Pseudomonas aeruginosa but unusual in that cytosine andguanine were used at nearly equal frequencies in the third codon position.The deduced P-protein product showed sequence homology with peptidesequences of the E. coli P-protein, the N-terminal portion of the E. coliT-protein (chorismate mutase:prephenate dehydrogenase), and themonofunctional prephenate dehydratases of Bacillus subtilis andCorynebacterium glutamicum. A narrow range of values (26-35%) for aminoacid matches revealed by pairwise alignments of monofunctional andbifunctional proteins possessing activity for prephenate dehydratasesuggests that extensive divergence has occurred between even the nearestphylogenetic lineages.

• Schmidheini T, Mosch HU, Evans JN, Braus G
• Yeast allosteric chorismate mutase is locked in the activated state by asingle amino acid substitution.
• Biochemistry. 1990; 29: 3660-8
• Display abstract

• Chorismate mutase, a branch-point enzyme in the aromatic amino acidpathway of Saccharomyces cerevisiae, and also a mutant chorismate mutasewith a single amino acid substitution in the C-terminal part of theprotein have been purified approximately 20-fold and 64-fold fromoverproducing strains, respectively. The wild-type enzyme is activated bytryptophan and subject to feedback inhibition by tyrosine, whereas themutant enzyme does not respond to activation by tryptophan nor inhibitionby tyrosine. Both enzymes are dimers consisting of two identical subunitsof Mr 30,000, each one capable of binding one substrate and one activatormolecule. Each subunit of the wild-type enzyme also binds one inhibitormolecule, whereas the mutant enzyme lost this ability. The enzyme reactionwas observed by 1H NMR and shows a direct and irreversible conversion ofchorismate to prephenate without the accumulation of any enzyme-freeintermediates. The kinetic data of the wild-type chorismate mutase showpositive cooperativity toward the substrate with a Hill coefficient of1.71 and a [S]0.5 value of 4.0 mM. In the presence of the activatortryptophan, the cooperativity is lost. The enzyme has an [S]0.5 value of1.2 mM in the presence of 10 microM tryptophan and an increased [S]0.5value of 8.6 mM in the presence of 300 microM tyrosine. In the mutantenzyme, a loss of cooperativity was observed, and [S]0.5 was reduced to1.0 mM. This enzyme is therefore locked in the activated state by a singleamino acid substitution.

• Ahmad S, Wilson AT, Jensen RA
• Chorismate mutase:prephenate dehydratase from Acinetobacter calcoaceticus.Purification, properties and immunological cross-reactivity.
• Eur J Biochem. 1988; 176: 69-79
• Display abstract

• The bifunctional P protein (chorismate mutase: prephenate dehydratase)from Acinetobacter calcoaceticus has been purified. It was homogeneous inpolyacrylamide gels and was more than 95% pure on the basis of theimmunostaining of purified P protein with the antibodies raised againstthe P protein. The native enzyme is a homodimer (Mr = 91,000) composed of45-kDa subunits. A twofold increase in the native molecular mass of the Pprotein occurred in the presence of L-phenylalanine (inhibitor of bothactivities) or L-tyrosine (activator of the dehydratase activity) duringgel filtration. Chorismate mutase activity followed Michaelis-Mentenkinetics with a Km of 0.55 mM for chorismate. L-Phenylalanine was arelatively poor non-competitive inhibitor of the mutase activity. Thechorismate mutase activity was also competitively inhibited by prephenate(reaction product). Substrate-saturation curves for the dehydrataseactivity were sigmoidal showing positive cooperativity among theprephenate-binding sites. L-Tyrosine activated prephenate dehydratasestrongly but did not abolish positive cooperativity with respect toprephenate. L-Phenylalanine inhibited the dehydratase activity, and thesubstrate-saturation curves became increasingly sigmoidal as phenylalanineconcentrations were increased with happ values changing from 2.0 (nophenylalanine) to 4.0 (0.08 mM L-phenylalanine). A sigmoidal inhibitioncurve of the dehydratase activity by L-phenylalanine gave Hill plotshaving a slope of -2.9. Higher ionic strength increased the dehydrataseactivity by reducing the positive cooperative binding of prephenate, andthe sigmoidal substrate-saturation curves were changed to near-hyperbolicform. The happ values decreased with increase in ionic strength.Antibodies raised against the purified P protein showed cross-reactivitywith the P proteins from near phylogenetic relatives of A. calcoaceticus.At a greater phylogenetic distance, cross-reaction was superior with Pprotein from Neisseria gonorrhoeae than with that from the more closelyrelated Escherichia coli.

• Berry A, Byng GS, Jensen RA
• Interconvertible molecular-weight forms of the bifunctional chorismatemutase-prephenate dehydratase from Acinetobacter calcoaceticus.
• Arch Biochem Biophys. 1985; 243: 470-9
• Display abstract

• Acinetobacter calcoaceticus belongs to a large phylogenetic cluster ofgram-negative procaryotes that all utilize a bifunctional P-protein(chorismate mutase-prephenate dehydratase) [EC 5.4.99.5-4.2.1.51] forphenylalanine biosynthesis. These two enzyme activities from Ac.calcoaceticus were inseparable by gel-filtration or DEAE-cellulosechromatography. The molecular weight of the P-protein in the absence ofeffectors was 65,000. In the presence of L-tyrosine (dehydrataseactivator) or L-phenylalanine (inhibitor of both P-protein activities),the molecular weight increased to 122,000. Maximal activation (23-fold) ofprephenate dehydratase was achieved at 0.85 mM L-tyrosine. Under theseconditions, dehydratase activity exhibited a hysteretic response toincreasing protein concentration. Substrate saturation curves forprephenate dehydratase were hyperbolic at L-tyrosine concentrationssufficient to give maximal activation (yielding a Km,app of 0.52 mM forprephenate), whereas at lower L-tyrosine concentrations the curves weresigmoidal. Dehydratase activity was inhibited by L-phenylalanine, andexhibited cooperative interactions for inhibitor binding. A Hill plotyielded an n' value of 3.1. Double-reciprocal plots of substratesaturation data obtained in the presence of L-phenylalanine indicatedcooperative interactions for prephenate in the presence of inhibitor. Then values obtained were 1.4 and 3.0 in the absence or presence of 0.3 mML-phenylalanine, respectively. The hysteretic response of chorismatemutase activity to increasing enzyme concentration was less dramatic thanthat of prephenate dehydratase. A Km,app for chorismate of 0.63 mM wasobtained. L-Tyrosine did not affect chorismate mutase activity, but mutaseactivity was inhibited both by L-phenylalanine and by prephenate.Interpretations are given about the physiological significance of theoverall pattern of allosteric control of the P-protein, and therelationship between this control and the effector-inducedmolecular-weight transitions. The properties of the P-protein inAcinetobacter are considered within the context of the ubiquity of theP-protein within the phylogenetic cluster to which this genus belongs.