The stable evolutionary fixation of a bifunctional tyrosine-pathway protein in enteric bacteria

Abstract The bifunctional T-protein (chorismate mutase-T: cyclohexadienyl dehydrogenase) of l -tyrosine biosynthesis was found to be present in all genera making up the enteric bacteria. The dehydrogenase component of the T-protein was active with both prephenate and l -arogenate, showing it to be a cyclohexadienyl dehydrogenase. The dehydrogenase component, but not the mutase component, of the T-protein was feedback-inhibited by l -tyrosine. Unlike some other bifunctional proteins, the T-protein has evolved recently and is not ubiquitous. However, once the biochemical specialization of bifunctionality becomes established, the results indicate that such character states are strongly conserved through evolutionary time. Thus, bifunctional proteins can provide particularly reliable markers for small (recent origin), intermediate, and large (ancient origin) phylogenetic clusters.     

A monofunctional prephenate dehydrogenase created by cleavage of the 5' 109 bp of the tyrA gene from Erwinia herbicola.

A cohesive phylogenetic cluster that is limited to enteric bacteria and a few closely related genera possesses a bifunctional protein that is known as the T-protein and is encoded by tyrA. The T-protein carries catalytic domains for chorismate mutase and for cyclohexadienyl dehydrogenase. Cyclohexadienyl dehydrogenase can utilize prephenate or L-arogenate as alternative substrates. A portion of the tyr A gene cloned from Erwinia herbicola was deleted in vitro with exonuclease III and fused in-frame with a 5' portion of lacZ to yield a new gene, denoted tyrA*, in which 37 N-terminal amino acids of the T-protein are replaced by 18 amino acids encoded by the polycloning site/5' portion of the lacZ alpha-peptide of pUC19. The TyrA* protein retained dehydrogenase activity but lacked mutase activity, thus demonstrating the separability of the two catalytic domains. While the Km of the TyrA* dehydrogenase for NAD+ remained unaltered, the Km for prephenate was fourfold greater and the Vmax was almost twofold greater than observed for the parental T-protein dehydrogenase. Activity with L-arogenate, normally a relatively poor substrate, was reduced to a negligible level. The prephenate dehydrogenase activity encoded by tyrA* was hypersensitive to feedback inhibition by L-tyrosine (a competitive inhibitor with respect to prephenate), partly because the affinity for prephenate was reduced and partly because the Ki value for L-tyrosine was decreased from 66 microM to 14 microM. Thus, excision of a portion of the chorismate mutase domain is shown to result in multiple extra-domain effects upon the cyclohexadienyl dehydrogenase domain of the bifunctional protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The prephenate dehydrogenase component of the bifunctional T-protein in enteric bacteria can utilize L-arogenate

The prephenate dehydrogenase component of the bifunctional T-protein (chorismate mutase:prephenate dehydrogenase) has been shown to utilize L-arogenate, a common precursor of phenylalanine and tyrosine in nature, as a substrate. Partially purified T-protein from Klebsiella pneumoniae and from Escherichia coli strains K 12, B, C and W was used to demonstrate the utilization of L-arogenate as an alternative substrate for prephenate in the presence of nicotinamide adenine dinucleotide as cofactor. The formation of L-tyrosine from L-arogenate by the T-protein dehydrogenase was confirmed by high-performance liquid chromatography. As expected of a common catalytic site, dehydrogenase activity with either prephenate or L-arogenate was highly sensitive to inhibition by L-tyrosine.     

Evolution of the regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase present in the Escherichia coli genealogy.

The evolutionary history of isozymes for 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase has been constructed in a phylogenetic cluster of procaryotes (superfamily B) that includes Escherichia coli. Members of superfamily B that have been positioned on a phylogenetic tree by oligonucleotide cataloging possess one or more of four distinct isozymes of DAHP synthase. DAHP synthase-0 is insensitive to feedback inhibition, while DAHP synthase-Tyr, DAHP synthase-Trp, and DAHP synthase-Phe are sensitive to feedback inhibition by L-tyrosine, L-tryptophan, and L-phenylalanine, respectively. The evolutionary history of this isozyme family can be deduced within superfamily B by using a cladistic methodology of maximum parsimony (R. A. Jensen, Mol. Biol. Evol. 2:92-108, 1985). DAHP synthase-0 was found in Acinetobacter species and in Oceanospirillum minutulum, organisms that also possess DAHP synthase-Tyr. These two isozymes were apparently present in a common ancestor that predated the evolutionary divergence of contemporary superfamily B sublineages. DAHP synthase-0 is postulated to have been the evolutionary forerunner of DAHP synthase-Trp. The newly evolved DAHP synthase-Trp is postulated to have possessed sensitivity to feedback inhibition by chorismate as well as by L-tryptophan, chorismate sensitivity having been retained in rRNA group I pseudomonads (minor sensitivity), group V pseudomonads (very sensitive), and Lysobacter enzymogenes (ultrasensitive). Organisms constituting the enteric lineage of the phylogenetic tree (including a cluster of four Oceanospirillum species) have all lost the chorismate sensitivity of DAHP synthase-Trp. The absence of DAHP synthase-Phe in the Oceanospirillum cluster of organisms supports the previous conclusion that DAHP synthase-Phe evolved recently within superfamily B, being present only Escherichia coli and its close relatives.     

基于结构域活性分析的大肠杆菌T蛋白结构研究

大肠杆菌T蛋白含有三个结构域:分支酸变位酶、预苯酸脱氢酶和调节结构域。文章作者分段克隆了T蛋白的分支酸变位酶、预苯酸脱氢酶和调节结构域等片段,并对其进行了活性研究。研究发现,定位于N末端的分支酸变位酶结构域的比活性虽然不高,而稳定性较好;同时拥有调节结构域和预苯酸脱氢酶结构域的C末端片段,其预苯酸脱氢酶比活性的剩余百分率虽然高于分支酸变位酶结构域,但稳定性较差。作者进而表达了C末端切除38个氨基酸的T/1-336片段,发现预苯酸脱氢酶活性彻底丧失,而其分支酸变位酶和调节结构域的活性却基本保留。这说明T蛋白中分支酸变位酶结构域拥有一个相对独立、完整的结构,而预苯酸脱氢酶结构域和调节结构域交织共存,结构松散。     

Phylogenetic distribution of components of the overflow pathway tol-phenylalanine within the enteric lineage of bacteria

The enteric lineage of prokaryotes (traditional enteric bacteria,Aeromonas, andAlteromonas) encompasses closely related genera that share many common character states of aromatic amino acid biosynthesis. For example, they uniformly employ the tightly regulated bifunctional P-protein (chorismate mutase: prephenate dehydratase) to forml-phenylalanine via phenylpyruvate. A second, unregulated pathway to phenylalanine, originally termed the overflow pathway inPseudomonas aeruginosa, consists of a monofunctional chorismate mutase (CM-F) and a cyclohexadienyl dehydratase. The evolution of the overflow pathway has been dynamic in the enteric lineage.Serratia marcescens, Erwinia herbicola, Erwinia amylovora, and several otherErwinia species possess an intact pathway.Salmonella, Klebsiella, andErwinia carotovora possess an incomplete overflow pathway, whileEscherichia, Proteus, Aeromonas, andAlteromonas lack it altogether.     

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.     

Probing the overlap of chorismate mutase and prephenate dehydrogenase sites in the escherichia coli T-protein: a dehydrogenase-selective inhibitor

An inhibitor of prephenate dehydrogenase has been identified that has no effect on the chorismate mutase activity in the Escherichia coli T-protein, thus supporting the idea of two separate active sites.     

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.     

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.     

大肠杆菌P蛋白和T蛋白的调节结构域互换研究

在细菌、真菌及植物中,分支酸是一种位于关键分叉点上的中间代谢物,是所有芳香族氨基酸合成的共同前体.它可在双功能酶分支酸变位酶(CM)和预苯酸脱水酶(PDT)的催化下合成苯丙氨酸,在另一个双功能酶分支酸变位酶和预苯酸脱氢酶(PDH)的催化下合成酪氨酸.前者被称为P蛋白,后者被称为T蛋白.大肠杆菌P蛋白和T蛋白有着类似的结构,P蛋白由CMp、PDT和调节结构域３个独立结构域组成,其变构调节因子是苯丙氨酸.T蛋白只有CMt和PDH两个独立结构域组成,起变构调节作用的调节结构域与PDH密不可分,其变构调节因子是酪氨酸.为了研究P蛋白和T蛋白的调节结构域的变构调节作用,应用融合蛋白技术将P蛋白和T蛋白的调节结构域进行了互换.结果发现,互换了的调节结构域仍然具有变构调节作用,而且调节结构域的互换导致了变构调节因子的互换,说明调节结构域对酶活性的调节作用是非专一的,而其R结构域与调节因子的结合却是专一的.     

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 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.     

A reporter system that discriminates EF-hand-sensor motifs from signal-modulators at the single-motif level

The T-protein is a single-polypeptide bi-functional enzyme composed of a chorismate mutase domain fused to a prephenate dehydrogenase domain (TyrA). We replaced the chorismate mutase domain with canonical or pseudo-Ca²⁺-binding motifs (EF-hand). Canonical-EF-hand-motifs differentiate from pseudo-EF-hand-motifs by experimenting a Ca²⁺-dependent conformational change. The Ca²⁺-free EF-hand-TyrA fusion-proteins showed TyrA activity at the T-protein level. Canonical-EF-hand-TyrA fusions showed a Ca²⁺-dependent loss of TyrA activity, but a pseudo-EF-hand-TyrA fusion showed high TyrA activity level in excess-Ca²⁺ conditions. Because TyrA activity exhibits robust changes in response to Ca²⁺-dependent-EF-hand conformational alterations, TyrA could be a good Ca²⁺-reporter enzyme. A chimeric canonical/pseudo-EF-hand strategy is proposed to confer pseudo-EF-hand motifs with a Ca²⁺-dependent conformational change.     

Enzyme Alterations in Tyrosine and Phenylalanine Auxotrophs of Salmonella typhimurium

The enzyme activities specified by the tyrA and pheA genes were studied in wildtype strain Salmonella typhimurium and in phenylalanine and tyrosine auxotrophs. As in Aerobacter aerogenes and Escherichia coli, the wild-type enzymes of Salmonella catalyze two consecutive reactions: chorismate --> prephenate --> 4-hydroxy-phenylpyruvate (tyrA), and chorismate --> prephenate --> phenylpyruvate (pheA). A group of tyrA mutants capable of interallelic complementation had altered enzymes which retained chorismate mutase T activity but lacked prephenate dehydrogenase. Similarly, pheA mutants (in which interallelic complementation does not occur) had one group with altered enzymes which retained chorismate mutase P but lacked prephenate dehydratase. Tyrosine and phenylalanine auxotrophs outside of these categories showed loss of both activities of their respective bifunctional enzyme. TyrA mutants which had mutase T were considerably derepressed in this activity by tyrosine starvation and consequently excreted prephenate. A new and specific procedure was developed for assaying prephenate dehydrogenase activity.     

Cloning, sequencing, and expression of the P-protein gene (pheA) of Pseudomonas stutzeri in Escherichia coli: implications for evolutionary relationships in phenylalanine biosynthesis

The pheA gene encoding the bifunctional P-protein (chorismate mutase:prephenate dehydratase) was cloned from Pseudomonas stutzeri and sequenced. This is the first gene of phenylalanine biosynthesis to be cloned and sequenced from Pseudomonas. The pheA gene was expressed in Escherichia 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 the native enzyme from P. stutzeri. The nucleotide sequence of the P. stutzeri pheA gene was 1095 base pairs in length, predicting a 365-residue protein product with an Mr of 40,844. Codon usage in the P. stutzeri pheA gene was similar to that of Pseudomonas aeruginosa but unusual in that cytosine and guanine were used at nearly equal frequencies in the third codon position. The deduced P-protein product showed sequence homology with peptide sequences of the E. coli P-protein, the N-terminal portion of the E. coli T-protein (chorismate mutase:prephenate dehydrogenase), and the monofunctional prephenate dehydratases of Bacillus subtilis and Corynebacterium glutamicum. A narrow range of values (26-35%) for amino acid matches revealed by pairwise alignments of monofunctional and bifunctional proteins possessing activity for prephenate dehydratase suggests that extensive divergence has occurred between even the nearest phylogenetic lineages.     

The recent evolutionary origin of the phenylalanine-sensitive isozyme of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in the enteric lineage of bacteria

Summary Evolutionary events that generated the three regulatory isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase present in contemporary strains ofEscherichia coli have been proposed recently [Ahmad et al. (1986) J Bacteriol 165:146–154]. The phylogenetic subdivision of gram-negative prokaryotes studied (Superfamily B) includes enteric bacteria, anOceanospirillum cluster, pseudomonad Group I (e.g.,Pseudomonas aeruginosa), pseudomonad Group V (e.g.,Xanthomonas), and theAcinetobacter grouping. DAHP synthase-phe, a regulatory isozyme subject to allosteric control byl-phenylalanine, was the last member of the isozyme family to evolve. Thus, DAHP synthase-phe is absent throughout Superfamily B except within the enteric lineage. Bacteria that make up the enteric lineage (Escherichia, Klebsiella, Erwinia, Serratia, Proteus, Aeromonas, andAlteromonas) were examined in detail; DAHP synthasephe was present in each of these organisms. Therefore, the isozyme originated between the separation of the enteric andOceanospirillum lineages, prior to the divergence ofAlteromonas putrefaciens (44% homology withE. coli by DNA:rRNA hybridization) from the rest of the enteric lineage. DAHP synthase-tyr and DAHP synthase-trp were uniformly present within the enteric lineage, although it was often necessary to derepress DAHP synthase-trp by physiological manipulation in order to demonstrate its presence.     

Crystal structure of prephenate dehydrogenase from Aquifex aeolicus. Insights into the catalytic mechanism

The enzyme prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. Prephenate dehydrogenases exist as either monofunctional or bifunctional enzymes. The bifunctional enzymes are diverse, since the prephenate dehydrogenase domain is associated with other enzymes, such as chorismate mutase and 3-phosphoskimate 1-carboxyvinyltransferase. We report the first crystal structure of a monofunctional prephenate dehydrogenase enzyme from the hyper-thermophile Aquifex aeolicus in complex with NAD+. This protein consists of two structural domains, a modified nucleotide-binding domain and a novel helical prephenate binding domain. The active site of prephenate dehydrogenase is formed at the domain interface and is shared between the subunits of the dimer. We infer from the structure that access to the active site is regulated via a gated mechanism, which is modulated by an ionic network involving a conserved arginine, Arg250. In addition, the crystal structure reveals for the first time the positions of a number of key catalytic residues and the identity of other active site residues that may participate in the reaction mechanism; these residues include Ser126 and Lys246 and the catalytic histidine, His147. Analysis of the structure further reveals that two secondary structure elements, beta3 and beta7, are missing in the prephenate dehydrogenase domain of the bifunctional chorismate mutase-prephenate dehydrogenase enzymes. This observation suggests that the two functional domains of chorismate mutase-prephenate dehydrogenase are interdependent and explains why these domains cannot be separated.     

Evolutionary implications of features of aromatic amino acid biosynthesis in the genus Acinetobacter

Key enzymes of aromatic amino acid biosynthesis were examined in the genus Acinetobacter. Members of this genus belong to a suprafamilial assemblage of Gram-negative bacteria (denoted Superfamily B) for which a phylogenetic tree based upon oligonucleotide cataloging of 16S rRNA exists. Since the Acinetobacter lineage diverged at an early evolutionary time from other lineages within Superfamily B, an examination of aromatic biosynthesis in members of this genus has supplied improtant clues for the deduction of major evolutionary events leading to the contemporary aromatic pathways that now exist within Superfamily B. Together with Escherichia coli, Pseudomonas aeruginosa and Xanthomonas campestris, four well-spaced lineages have now been studied in comprehensive detail with respect to comparative enzymological features of aromatic amino acid biosynthesis. A. calcoaceticus and A. lwoffii both possess two chorismate mutase isozymes: one a monofunctional isozyme (chorismate mutase-F), and the other (chorismate mutase-P) a component of a bifunctional P-protein (chorismate mutase-prephenate dehydratase). While both P-protein activities were feedback inhibited by l-phenylalanine, the chorismate mutase-P activity was additionally inhibited by prephenate. Likewise, chorismate mutase-F was product inhibited by prephenate. Two isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase were detected. The major isozyme (>95%) was sensitive to feedback inhibition by l-tyrosine, whereas the minor isozyme was apparently insensitive to allosteric control. Prephenate dehydrogenase and arogenate dehydrogenase activities were both detected, but could not be chromatographically resolved. Available evidence favors the existence of a single dehydrogenase enzyme, exhibiting substrate ambiguity for prephenate andl-arogenate. Dehydrogenase activity with either of the latter substrates was specific for NADP⁺, NAD⁺ being ineffective. Consideration of the phylogeny of Superfamily-B organisms suggests that the stem ancestor of the Superfamily possessed a single dehydrogenase enzyme having ambiguity for both substrate and pyridine nucleotide cofactor. Since all other members of Superfamily B have NAD⁺-specific dehydrogenases, specialization for NADP⁺ must have occurred following the point of Acinetobacter divergence, leading to the dichotomy seen in present-day Superfamily-B organisms.     

Absolute dependence of phenylalanine and tyrosine biosynthetic enzyme on tryptophan in Candida maltosa

Candida maltosa synthesizes phenylalanine and tyrosine only via phenylpyruvate and p-hydroxyphenylpyruvate. Tryptophan is absolutely necessary for the enzymatic reaction of chorismate mutase and prephenate dehydrogenase; activity of prephenate dehydratase can be increased 2.5-fold in the presence of tryptophan. Activation of the chorismate mutase, prephenate dehydratase and prephenate dehydrogenase by tryptophan is competitive with respect to chorismate and prephenate with Ka 0.06mM, 0.56mM and 1.7mM. In addition tyrosine is a competitive inhibitor of chorismate mutase (Ki = 0.55mM) and prephenate dehydrogenase (Ki = 5.5mM).