Regulation of a Ligand-Mediated Association-Dissociation System of Anthranilate Synthesis in Clostridium butyricum

The anthranilate synthetase of Clostridium butyricum is composed of two nonidentical subunits of unequal size. An enzyme complex consisting of both subunits is required for glutamine utilization in the formation of anthranilic acid. Formation of anthranilate will proceed in the presence of partially pure subunit I provided ammonia is available in place of glutamine. Partially pure subunit II neither catalyzes the formation of anthranilate nor possesses anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity. The enzyme complex is stabilized by high subunit concentrations and by the presence of glutamine. High KCl concentrations promote dissociation of the enzyme into its component subunits. The synthesis of subunits I and II is coordinately controlled with the synthesis of the enzymes mediating reactions 4 and 5 of the tryptophan pathway. When using gel filtration procedures, the molecular weights of the large (I) and small (II) subunits were estimated to be 127,000 and 15,000, respectively. Partially pure anthranilate synthetase subunits were obtained from two spontaneous mutants resistant to growth inhibition by 5-methyltryptophan. One mutant, strain mtr-8, possessed an anthranilate synthetase that was resistant to feedback inhibition by tryptophan and by three tryptophan analogues: 5-methyl-tryptophan, 4- and 5-fluorotryptophan. Reconstruction experiments carried out by using partially purified enzyme subunits obtained from wild-type, mutant mtr-8 and mutant mtr-4 cells indicate that resistance of the enzyme from mutant mtr-8 to feedback inhibition by tryptophan or its analogues was the result of an alteration in the large (I) subunit. Mutant mtr-8 incorporates [(14)C]tryptophan into cell protein at a rate comparable with wild-type cells. Mutant mtr-4 failed to incorporate significant amounts of [(14)C]tryptophan into cell protein. We conclude that strain mtr-4 is resistant to growth inhibition by 5-methyltryptophan because it fails to transport the analogue into the cell. Although mutant mtr-8 was isolated as a spontaneous mutant having two different properties (altered regulatory properties and an anthranilate synthetase with altered sensitivity to feedback inhibition), we have no direct evidence that this was the result of a single mutational event.     

Tryptophan Synthetic Pathway and Its Regulation in Chromobacterium violaceum

Extracts of Chromobacterium violaceum catalyzed all of the reactions involved in synthesizing tryptophan from chorismic acid. Tryptophan auxotrophs which had lost any of these activities did not produce the characteristic purple pigment, violacein, when grown on a medium in which tryptophan was limiting. Gel filtration of extracts allowed us to estimate molecular weights for the tryptophan enzymes. All of the enzymes appeared to have molecular weights below 100,000. No enzymes were observed to occur in aggregates. The specific activities of the enzymes of the tryptophan pathway did not change when mutants were grown under conditions of limiting or excess tryptophan. The first enzyme in the pathway, anthranilate synthetase, was subject to feedback control by the end product, tryptophan. Tryptophan acted as a noncompetitive inhibitor with respect to glutamine, one of the substrates for anthranilate synthetase, and as a competitive inhibitor of the reaction when chorismate, the other substrate, was varied. The nonlinearity observed in the Lineweaver-Burk plot in the latter case suggests that there may be more than one chorismate-binding site on anthranilate synthetase.     

Enzymatic Basis for Overproduction of Tryptophan and Its Metabolites in Hansenula polymorpha Mutants

3-Deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthetase and anthranilate synthetase are key regulatory enzymes in the aromatic amino acid biosynthetic pathway. The DAHP synthetase activity of Hansenula polymorpha was subject to additive feedback inhibition by phenylalanine and tyrosine but not by tryptophan. The synthesis of DAHP synthetase in this yeast was not repressed by exogenous aromatic amino acids, singly or in combinations. The activity of anthranilate synthetase was sensitive to feedback inhibition by tryptophan, but exogenous tryptophan did not repress the synthesis of this enzyme. Nevertheless, internal repression of anthranilate synthetase probably exists, since the content of this enzyme in H. polymorpha strain 3-136 was double that in the wild-type and less sensitive 5-fluorotryptophan-resistant strains. The biochemical mechanism for the overproduction of indoles by the 5-fluorotryptophan-resistant mutants was due primarily to a partial desensitization of the anthranilate synthetase of these strains to feedback inhibition by tryptophan. These results support the concept that inhibition of enzyme activities rather than enzyme repression is more important in the regulation of aromatic amino acid biosynthesis in H. polymorpha.     

Enzymes of the Tryptophan Pathway in Acinetobacter calco-aceticus

All enzymes of the tryptophan synthetic pathway were detectable in extracts from wild-type Acinetobacter calco-aceticus. The levels of these enzymes were determined in extracts from a number of auxotrophs grown under limiting tryptophan. In each case only anthranilate synthetase was found to be present in increased amounts, whereas the specific activities of the remaining enzymes remained unchanged and unaffected by the tryptophan concentration. Derepression of anthranilate synthetase was found to occur as the concentration of tryptophan became limiting. Anthranilate synthetase and phosphoribosyl transferase activities are both feedback-inhibited by tryptophan. Molecular weight determination carried out by gel filtration and zonal centrifugation in sucrose revealed that all the enzymes are less than 100,000, and no molecular aggregates of these enzymes were detected. The data indicate that tryptophan synthesis in Acinetobacter is regulated both by feedback inhibition of the first two enzymes of the pathway and by repression control of anthranilate synthetase.     

The Arabidopsis thaliana trp5 mutant has a feedback-resistant anthranilate synthase and elevated soluble tryptophan.

The first step of tryptophan biosynthesis is catalyzed by anthranilate synthase (AS), which is normally subject to feedback inhibition by tryptophan. Three independent trp5 mutants defective in the Arabidopsis thaliana AS alpha subunit structural gene ASA1 were identified by selection for resistance to the herbicidal compound 6-methylanthranilate. In all three mutants these biochemical changes are caused by a single amino acid substitution from aspartate to asparagine at residue position 341. Compared with the enzyme from wild-type plants, the tryptophan concentration causing 50% inhibition of AS activity in the trp5 mutant increased nearly 3-fold, the apparent Km for chorismate decreased by approximately 50%, and the apparent Vmax increased 60%. As a consequence of altered AS kinetic properties, the trp5 mutants accumulated 3-fold higher soluble tryptophan than wild-type plants. However, even though the soluble tryptophan levels were increased in trp5 plants, the concentrations of five tryptophan biosynthetic proteins remained unchanged. These data are consistent with the hypothesis that the reaction catalyzed by A. thaliana AS is rate limiting for the tryptophan pathway and that accumulation of tryptophan biosynthetic enzymes is not repressed by a 3-fold excess of end product.     

Characterization of a 5-methyltryptophan resistant strain of Catharanthus roseus cultured cells

Strains of Catharanthus roseus suspension cells resistant to growth inhibition by various tryptophan analogs were selected. Tryptophan synthetase and anthranilate synthetase from the resistant cells differed from the normal cell enzymes by being more resistant to feedback inhibition by tryptophan. Though these altered enzymes allowed the free tryptophan level of the resistant cells to be 3–40 times higher than that of normal cells, the accumulation of tryptamine or ajmalicine could not be detected in the resistant cells.     

Evidence for Compartmentation of Tryptophan in Cultured Plant Tissues: Free Tryptophan Levels and Inhibition of Anthranilate Synthetase

1. Anthranilate synthetase activity in crude extracts from tissue cultures of Daucus carota L. (carrot), Nicotiana tabacum L. (tobacco; cv. Wisconsin 38 and xanthi), Glycine max Merr. (soybean) and Oryza sativa L. (rice) was completely inhibited by l -tryptophan (5 to 50 μM). Mutant carrot and tobacco lines, capable of growth in the presence of 5-methyltryptophan, required 500 to more than 1000 μM tryptophan for complete inhibition of enzyme activity, respectively. 2. Except for the mutant tobacco line, the concentrations of free tryptophan in all tissue cultures tested were greater than the levels necessary to completely inhibit the respective anthranilate synthetase activities in vitro. These findings would indicate that much of the free tryptophan is compartmentalized away from the regulatory enzyme, anthranilate synthetase. This could implicate compartmentalization of the inhibitor as a biosynthetic control mechanism. 3. During the growth of normal and mutant carrot tissues the anthranilate synthetase enzyme must be at least 7.8 and 10.8% active, respectively, in order to accumulate the amount of tryptophan found in the tissues. 4. Of the substrates and cofactors required for anthranilate synthetase activity in vitro, Mg²⁺ and glutamine were present at near optimal levels in the carrot and tobacco tissues, but chorismate was found to be significantly below the optimal concentrations.     

Anthranilate synthase/anthranilate 5-phosphoribosyl 1-pyrophosphate phosphoribosyltransferase from Aerobacter aerogenes

1. Anthranilate synthase and phosphoribosyltransferase from Aerobacter aerogenes purify simultaneously and sediment together on sucrose gradients, showing that they occur as an enzyme aggregate. Both activities of the intact aggregate are subject to inhibition by tryptophan. 2. By using appropriate auxotrophic mutants it was shown that an intact active enzyme aggregate is formed when the components come from separate mutant strains. An intact active aggregate can also be formed when one component is from Escherichia coli and the other from A. aerogenes. 3. Phosphoribosyltransferase of A. aerogenes is active when not in an aggregate with anthranilate synthase, but is not subject to tryptophan inhibition, indicating that the inhibitor site is on the anthranilate synthase component. 4. Anthranilate synthase can be active and sensitive to tryptophan inhibition when complexed with an inactive phosphoribosyltransferase. 5. Kinetic studies on the anthranilate synthase activity show that tryptophan is a competitive inhibitor with respect to chorismate and a non-competitive inhibitor with respect to either glutamine or NH(4) (+) ions. This is consistent with a sequential mechanism of the ordered type in which chorismate is the first reactant.     

Regulation of the chorismic acid branch-point in aromatic acid synthesis in blue-green and green algae

Summary In extension of previous studies on the regulation of the aromatic amino acid pathway in blue-green and green algae the control of two branch-point enzymes, namely chorismate mutase and anthranilate synthetase has been studied. The activity of chorismate mutase in these organisms is effectively inhibited by l-tyrosine or l-phenylalanine. l-tryptophan, in contrast, proved to be a positive effector of the enzyme: in the absence of phenylalanine or tyrosine tryptophan slightly stimulated chorismate mutase activity; this stimulation was even brought about in the presence of excess phenylalanine or tyrosine, irrespective if the enzyme had been preincubated with these inhibitors or not. Tryptophan thus proved to completely revert the feedback inhibition of this enzyme by phenylalanine or tyrosine. Substrate saturation curves of chorismate mutase activity are hyperbolic in the presence of tryptophan and sigmoid in the presence of phenylalanine or tyrosine. In contrast to the enzymes of the green algae investigated, chorismate mutase activity of Anacystis nidulans, a member of the class of the blue-green algae was not affected by any of the aromatic amino acids.The activity of anthranilate synthetase, the second enzyme of the chorismic acid branch-point of the pathway was consistently inhibited by l-tryptophan in all the organisms tested. The results described here bear significance on the regulation of a multi-branched pathway the first enzyme of which is inhibited just by one endproduct.     

Control of Tryptophan Biosynthesis in Plant Tissue Cultures: Lack of Repression of Anthranilate and Tryptophan Synthetases by Tryptophan

Tobacco (cv. Xanthi and cv. Wisconsin 38), rice, carrot, tomato, and soybean tissue cultures were grown in liquid media containing L-tryptophan. The addition of tryptophan increased the cellular tryptophan levels greatly (12–2500 fold), but did not lower appreciably the levels of two tryptophan biosynthetic enzymes, anthranilate synthetase and tryptophan synthetase. However, the addition of 50 μM tryptophan to the crude enzyme extract completely inhibited the anthranilate synthetase activity while 1 mM tryptophan inhibited the tryptophan synthetase activity by only 10–20°/o. This information indicates that tryptophan biosynthesis is controlled by the feedback inhibition of anthranilate synthetase by tryptophan and not by repression of enzyme synthesis. All of the species had significant enzyme levels. Anthranilate synthetase activity could not be detected in extracts from cells grown on tryptophan unless the extracts were first passed through two G-25 Sephadex columns with a short 30 °C warming step in between, a procedure shown to remove an inhibitor of the enzyme.     

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.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

Molecular basis of alpha-methyltryptophan resistance in amt-1, a mutant of Arabidopsis thaliana with altered tryptophan metabolism.

A mutant of Arabidopsis thaliana, amt-1, was previously selected for resistance to growth inhibition by the tryptophan analog alpha-methyltryptophan. This mutant had elevated tryptophan levels and exhibited higher anthranilate synthase (AS) activity that showed increased resistance to feedback inhibition by tryptophan. In this study, extracts of the mutant callus exhibited higher AS activity than wild-type callus when assayed with either glutamine or ammonium sulfate as amino donor, thus suggesting that elevated AS activity in the mutant was due to an alteration in the alpha subunit of the enzyme. The mutant also showed cross-resistance to 5-methylanthranilate and 6-methylanthranilate and mapped to chromosome V at or close to ASA1 (a gene encoding the AS alpha subunit). ASA1 mRNA and protein levels were similar in mutant and wild-type leaf extracts. Levels of ASA1 mRNA and protein were also similar in callus cultures of mutant and wild type, although the levels in callus were higher than in leaf tissue. Sequencing of the ASA1 gene from amt-1 revealed a G to A transition relative to the wild-type gene that would result in the substitution of an asparagine residue in place of aspartic acid at position 341 in the predicted amino acid sequence of the ASA1 protein. The mutant allele in strain amt-1 has been renamed trp5-1.     

Separation of Two Forms of Anthranilate Synthetase from 5-Methyltryptophar Susceptible and -Resistant Cultured Solarium tuberosumCells

Potato cell suspension cultures (Solanum tuberosumL. cv. Merrimack) have been selected which are resistant to growth inhibition by D,L-5-methyltryptophan. Anthranilate synthetase activity in crude extracts from resistant cells was less sensitive to feedback inhibition by L-tryptophan and D,L-5-methyltryptophan than the activity from the sensitive line. This altered feedback control apparently accounts for the cell's resistance to growth inhibition since there is a 48-fold increase in free tryptophan in one of the resistant cell lines. Preparative polyacrylamide gel electro-phoresis separated feedback-sensitive and -resistant forms of anthranilate synthetase in extracts from both 5-methyltryptophan-susceptible and -resistant cells, with a predominance of the corresponding form in the respective cell type. The anthranilate synthetase activity from the 5-methyltryptophan-resistant line was inactivated more slowly by incubation of crude extracts at 50°C than the activity from the sensitive line. These results suggest the presence of two isoenzymes of anthranilate synthetase in cultured potato cells.     

Isolation of cyanobacterial auxotrophs by direct selection of regulatory-mutant phenotypes

We have isolated a chorismate mutase bradytroph (leaky auxotroph) ofAnabaena sp. PCC 7119 (ATCC 29151) as a spontaneous 6-fluorotryptophan-resistant mutant. The decreased chorismate mutase activity resulted in the production of quantities of the phenylalanine and tyrosine that limited rate of growth. 3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity in the mutant was elevated more than twofold over the wild-type activity, suggesting derepression of this enzyme. The physiological deregulation of DAHP synthase and the genetic-based deficiency of chorismate mutase promoted an elevated level of intracellular chorismate, which then overwhelmed the competitive inhibition of anthranilate synthase by tryptophan, resulting in the overproduction of tryptophan and indoleglycerolphosphate. The presence of exogenous serine increased the production of tryptophan at the expense of indoleglycerolphosphate. This indicated that the endogenous potential for increasing the amount of serine available for increased tryptophan production is limited.     

Study of anthranilate synthase function by replacement of cysteine 84 using site-directed mutagenesis

Cysteine 84 was replaced by glycine in Serratia marcescens anthranilate synthase Component II using site-directed mutagenesis of cloned trpG. This replacement abolished the glutamine-dependent anthranilate synthase activity but not the NH3-dependent activity of the enzyme. The mutation provides further evidence for the role of active site cysteine 84 in the glutamine amide transfer function of anthranilate synthase Component II. By the criteria of circular dichroism, proteolytic inactivation, and feedback inhibition the mutant and wild type enzymes were structurally similar. The NH3-dependent anthranilate synthase activity of the mutant enzyme supported tryptophan synthesis in media containing a high concentration of ammonium ion.     

Anthranilate synthetase, an enzyme specified by the tryptophan operon of Escherichia coli: Comparative studies on the complex and the subunits

The properties of the anthranilate synthetase complex and its separated subunits were compared in catalyzing the anthranilate synthetase reaction, chorismate + l-glutamine or NH(4) (+) --> anthranilate, and the transferase reaction, anthranilate + 5'-phosphorylribosyl-1-pyrophosphate --> phosphoribosyl anthranilate. It is shown that anthranilate synthetase component I is activated by normal anthranilate synthetase component II, a component II(CRM) (CRM = immunologically cross-reacting material), and by a presumed fragment of component II produced by a deletion mutant. Significant differences between the complex and its subunits are demonstrated with respect to substrate affinity, thermostability, feedback inhibitor sensitivity, and activity in the presence of various divalent cations. Of particular interest are the findings that the transferase activity of component II is only inhibitable by l-tryptophan when the component is in the complex and that this inhibition does not appear to depend upon the feedback-sensitive site of component I.     

Regulation of the Escherichia coli tryptophan operon by early reactions in the aromatic pathway

7-Methyltryptophan (7MT) or compounds which can be metabolized to 7MT, 3-methylanthranilic acid (3MA) and 7-methylindole, cause derepression of the trp operon through feedback inhibition of anthranilate synthetase. Tyrosine reverses 3MA or 7-methylindole derepression, apparently by increasing the amount of chorismic acid available to the tryptophan pathway. A mutant isolated on the basis of 3MA resistance (MAR 13) was found to excrete small amounts of chorismic acid and to have a feedback-resistant phenylalanine 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. Genetic evidence indicates that the mutation conferring 3MA resistance and feedback resistance is very closely linked to aroG, the structural gene for the DAHP synthetase (phe). Since feedback inhibition of anthranilate synthetase by l-tryptophan (or 7MT) is competitive with chorismic acid, alterations in growth conditions (added tyrosine) or in a mutant (MAR 13) which increase the amount of chorismic acid available to the tryptophan pathway result in resistance to 7MT derepression. Owing to this competitive nature of tryptophan feedback inhibition of anthranilate synthetase by chorismic acid, the early pathway apparently serves to exert a regulatory influence on tryptophan biosynthesis.     

Anthranilate synthetase, an enzyme specified by the tryptophan operon of Escherichia coli: purification and characterization of component I

A procedure employed in the purification of anthranilate synthetase component I of Escherichia coli is described. The purified component appears homogeneous by starch gel electrophoresis and by sedimentation analysis. A molecular weight of 60,000 was estimated by gel filtration of Sephadex G-100. This value is consistent with the molecular weight estimated from the sedimentation and diffusion coefficients. Purified anthranilate synthetase component I cannot use glutamine as substrate and thus has no activity in the reaction of chorismate + l-glutamine --> anthranilate; however, it is active when ammonium sulfate is provided as amino donor. Sucrose density gradient analyses showed that ammonium sulfate does not affect the sedimentation velocity of component I. The ultraviolet absorption and fluorescence spectra of the purified component indicated that it contains tryptophan. Peptide pattern and extract complementation evidence suggested that the protein is a single polypeptide chain. Enzyme activity measurements indicated that wild-type E. coli produces equimolar amounts of at least four of the five polypeptides specified by the operon. Purified anthranilate synthetase component I is inhibited by l-tryptophan.     

Amino Acid Composition of Elm Seeds

In the biosynthetic pathway of aromatic amino acids of Brevibacterium flavum, ratios of each biosynthetic flow at the chorismate branch point were calculated from the reaction velocities of anthranilate synthetase for tryptophan and chorismate mutase for phenylalanine and tyrosine at steady state concentrations of chorismate. When these aromatic amino acids were absent, the ratio was 61, showing an extremely preferential synthesis of tryptophan. The presence of tryptophan at 0.01 mM decreased the ratio to 0.07, showing a diversion of the preferential synthesis to phenylalanine and tyrosine. Complete recovery by glutamate of the ability to synthesize the Millon-positive substance in dialyzed cell extracts confirmed that tyrosine was synthesized via pretyrosine in this organism. Partially purified prephenate aminotransferase, the first enzyme in the tyrosine-specific branch, had a pH optimum of 8.0 and Km’s of 0.45 and 22 mM for prephenate and glutamate, respectively, and its activity was increased 15-fold by pyridoxal-5-phosphate. Neither its activity nor its synthesis was affected at all by the presence of the end product tyrosine or other aromatic amino acids. The ratio of each biosynthetic flow for tyrosine and phenylalanine at the prephenate branch point was calculated from the kinetic equations of prephenate aminotransferase and prephenate dehydratase, the first enzyme in the phenylalanine-specific branch. It showed that tyrosine was synthesized in preference to phenylalanine when phenylalanine and tyrosine were absent. Furthermore, this preferential synthesis was diverted to a balanced synthesis of phenylalanine and tyrosine through activation of prephenate dehydratase by the tyrosine thus synthesized. The feedback inhibition of prephenate dehydratase by phenylalanine was proposed to play a role in maintaining a balanced synthesis when supply of prephenate was decreased by feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP*) synthetase, the common key enzyme. Overproduction of the end products in various regulatory mutants was also explained by these results.