Prephenate Dehydratase Activity Research Articles

A single cyclohexadienyl dehydratase specifies the prephenate dehydratase and arogenate dehydratase components of one of two independent pathways to l-phenylalanine in Erwinia herbicola

Dual biosynthetic pathways diverge from prephenate to l-phenylalanine in Erwinia herbicola, the unique intermediates of these pathways being phenylpyruvate and l-arogenate. After separation from the bifunctional P-protein (one component of which has prephenate dehydratase activity), the remaining prephenate dehydratase activity could not be separated from arogenate dehydratase activity throughout fractionation steps yielding a purification of more than 1200-fold. The ratio of activities was constant after removal of the P-protein, and the two dehydratase activities were stable during purification. Hence, the enzyme is a cyclohexadienyl dehydratase. The native enzyme has a molecular mass of 73 kDa and is a tetramer made up of identical 18-kDa subunits. K m values of 0.17 m m and 0.09 m m were calculated for prephenate and l-arogenate, respectively. l-Arogenate inhibited prephenate dehydratase competitively with respect to prephenate, whereas prephenate inhibited arogenate dehydratase competitively with respect to l-arogenate. Thus, the enzyme has a common catalytic site for utilization of prephenate or l-arogenate as alternative substrates. This is the first characterization of a purified monofunctional cyclohexadienyl dehydratase.

Biosynthesis of aromatic amino acids in Nocardia sp. 239: effects of amino acid analogues on growth and regulatory enzymes

Further steps required for overproduction of aromatic amino acids by a mutant strain of Nocardia sp. 239 (Noc 87-13), unable to grow on l-phenylalanine as a sole carbon and energy source, were investigated. A number of analogues of the aromatic amino acids displayed severe inhibitory effects on the activities of regulatory enzymes in the biosynthetic pathway and growth of the organism in glucose mineral medium. l-Tryptophane analogues strongly inhibited 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity. l-Tyrosine analogues especially inhibited DAHP synthase and chorismate mutase, whereas l-phenylalanine analogues strongly inhibited chorismate mutase and prephenate dehydratase activity. Addition of the aromatic amino acids and their precursors chorismate, 4-hydroxyphenylpyruvate, phenylpyruvate and anthranilate, to the medium counteracted the growth inhibitory effect of specific analogues. The data indicate that ortho- (OFP) and para-fluoro-d,l-phenylalanine (PFP), and l-phenylalanine amide, are the most suitable analogues for the isolation of feedback-inhibition-insensitive prephenate dehydratase mutants. Attempts to isolate l-tyrosine and l-trytophane auxotrophic mutants were only successful in the latter case, resulting in the selection of a stable anthranilate synthase-negative mutant (Noc 87-13-14). Uptake of aromatic amino acids in Nocardia sp. 239 most likely involves a common transport system. This necessitates the use of anthranilate, rather than l-trytophane, as a supplement during the isolation of l-tyrosine auxotrophic and OFP- and/or PFP-resistant mutant derivative strains of Noc 87-13-14.

Regulation of the biosynthetic pathway of aromatic amino acids in Nocardia mediterranei

The regulation of enzymes in the biosynthetic pathway of aromatic amino acids in Nocardia mediterranei was studied. Anthranilate synthase was sensitive to feedback inhibition by very low concentrations of lTrp, and kinetic analysis showed that lTrp was competitive with respect to chorismate; the five enzymes in lTrp biosynthesis pathway, anthranilate synthase (AS), anthranilate-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT), N-5′-phosphoribosylanthranilate isomerase (PRAI), indole-3-glycerol phosphate synthetase (InGPS) and tryptophan synthase (TS), were all repressed by lTrp; lTyr and lPhe inhibited chorismate mutase. Prephenate dehydratase activity was greatly inhibited by lPhe and activated by lTyr, nearly 60% of its activity was inhibited by 5 μM of lPhe, and 20μM of lTyr increased the activity approx. 3-fold. In addition, the effects of lPhe and lTyr on prephenate dehydratase were highly specific. The regulatory circuit of the biosynthetic pathway of aromatic amino acids in N. mediterranei is presented.

Regulation of metabolic branch points of aromatic amino acid biosynthesis in Pichia guilliermondii.

The regulatory properties of the enzymes involved in the aromatic amino acid biosynthesis of Pichia guilliermondii were investigated and compared with the regulatory pattern found in other yeast species. 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, anthranilate synthase, chorismate mutase and prephenate dehydrogenase are key regulatory enzymes in P. guilliermondii. Two distinctly regulated isozymes of DAHP synthase, the initial pathway enzyme, which is inhibited by tyrosine or phenylalanine were separated by DEAE-cellulose chromatography and were characterized. Tryptophan is an excellent feedback inhibitor of anthranilate synthase, the first definite step in tryptophan biosynthesis. There are two controlled enzymes within the specific synthesis of phenylalanine and tyrosine, chorismate mutase and prephenate dehydrogenase. Chorismate mutase exhibits a balanced allosteric responsivity to phenylalanine and tyrosine, when these are used as inhibitor; tryptophan acts as an allosteric activator. Tyrosine is an effective inhibitor of prephenate dehydrogenase, whereas the activity of prephenate dehydratase is not affected by any of the aromatic amino acids. The synthesis of the enzymes in the yeast was not repressed by any single exogenous aromatic amino acids, nor by combinations of the same.

The reactivity of the sulphydryl groups of chorismate mutase/prephenate dehydratase - a bifunctional enzyme of phynylalanine biosynthesis in Escherichia coli K12

The reaction of N-ethylmaleimide with chorismate mutase/prephenate dehydratase (chorismate pyruvatemutase/prephenate hydrolyase (decarboxylating) EC 5.4.99.5/EC 4.2.1.51) from Escherichia coli K12, which leads to the preferential inactivation of the prephenate dehydratase activity (Gething, M-J.H. and Davidson B.E. (1977) Eur. J. Biochem. 78, 111–117), was found to involve only the sulphydryl groups of the enzyme. Determination of the reactivities of the four different cysteine residues indicated that the reaction was not specific for a single residue, although two residues (Cys-216 and Cys-374) were more reactive than the others. The amount of inhibition of the prephenate dehydratase activity approximated in extent to the sum of the stoichiometries of the individual reactions of N-ethylmaleinide with these two cysteine residues. In the presence of either phenylpyruvate, the product of the prephenate dehydratase activity, or cis-aconitate, a competitive inhibitor with respect to prephenate, the prephenate dehydratase activity was substantially protected from inactivation. This protection was concomitant with a significant decline in the reactivities of both Cys-216 and Cys-374. These results are interpreted as indicating that both of these cysteine residues are at, or near to, the prephenate dehydratase active site and are possibly essential for the prephenate dehydratase activity of the enzyme.

Regulation of chorismate mutase, prephenate dehydrogenase and prephenate dehydratase of Candida maltosa

AbstractThe enzymes of the phenylalanine‐tyrosine pathway were partially purified from Candida maltosa and the regulatory patterns were established. Chorismate mutase (Mr 63,000), prephenate dehydrogenase (Mr 75,000) and prephenate dehydratase (Mr 88,000) were separated from each other. The formation of chorismate mutase was only regulated by a general control of amino acid biosynthesis, whereas the synthesis of prephenate dehydrogenase and prephenate dehydratase was constitutive. Both chorismate mutase and prephenate dehydrogenase were activated by tryptophan and by methylated or fluorinated tryptophan analogues. l‐tyrosine as well as α‐methyl‐dl‐tyrosine inhibited the activity of both enzymes. Prephenate dehydratase activity was stimulated by l‐tryptophan and by methylated tryptophan derivatives and inhibited by d‐tryptophan or 5‐fluorotryptophan. In contrast to chorismate mutase, the double‐reciprocal curves of substrate saturation of which were hyperbolical (positive cooperativity), prephenate dehydrogenase and prephenate dehydratase showed linear curves of substrate saturation, indicating normal Mi‐chaelis kinetics.

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).

A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K12 defective in the PheA gene

Three classes of mutant strains of Escherichia coli K12 defective in pheA, the gene coding for chorismate mutase/prephenate dehydratase, have been isolated: (1) those lacking prephenate dehydratase activity, (2) those lacking chorismate mutase activity, and (3) those lacking both activities. Chorismate mutase/prephenate dehydratase from the second class of mutants was less sensitive to inhibition by phenylalanine than wild-type enzyme and, along with the defective enzyme from the third class of mutants, could not be purified by affinity chromatography on Sepharosyl-phenylalanine. Pure chorismate mutase/prephenate dehydratase protein was prepared from two strains belonging to the first class. The chorismate mutase activity of these enzymes is kinetically similar to that of the wild-type enzyme except for a two- to threefold increase in both the K a for chorismate and the K is for inhibition by prephenate. In both cases only one change in the tryptic fingerprint was detected, resulting from a substitution of the threonine residue in the peptide Gln·Asn·Phe·Thr·Arg. This suggests that this residue is catalytically or structurally essential for the dehydratase activity.

Regulation of prephenate dehydratase in coryneform species of bacteria by l-phenylalanine and by remote effectors

Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes utilize the phenylpyruvate branchlet as the sole means of l-phenylalanine biosynthesis. Enzymological aspects of l-phenylalanine biosynthesis were generally similar in the three microbial species selected to represent the coryneform grouping. Thus, the general picture of aromatic biosynthesis in coryneform bacteria is very similar to that found in species of bluegreen algae, i.e., phenylpyruvate branchlet for l-phenylalanine biosynthesis and pretyrosine branchlet for l-tyrosine biosynthesis ( A. Fazel and R. A. Jensen, 1979, J. Bacteriol. 138, 805–815). Prephenate dehydratase is subject to repression control and to feedback inhibition by l-phenylalanine ( K i value range: 35–95 μ m). Extrapathway effectors activated ( l-tyrosine) or inhibited ( l-tryptophan) prephenate dehydratase, reminiscent of the action of remote effectors with Bacillus subtilis prephenate dehydratase. A labile activation of prephenate dehydratase by l-methionine was also found in C. glutamicum. Prephenate dehydratase copurifies with an aromatic aminotransferase which is capable of transaminating phenylpyruvate, 4-hydroxyphenylpyruvate, or prephenate in vitro. The possibility of a multifunctional protein was ruled out, however, by results obtained with l-phenylalanine auxotrophs lacking prephenate dehydratase and a regulatory mutant constitutive for prephenate dehydratase. Other classes of regulatory mutants altered in prephenate dehydratase lost sensitivity to inhibition by l-phenylalanine (and l-tryptophan), or produced an enzyme that was hypersensitive to l-tyrosine activation. Molecular weight estimates of the prephenate dehydratase enzymes isolated from the three species studied ranged from 200,000–260,000. Unlike the B. subtilis enzyme, neither allosteric (positive or negative) effectors nor regulatory mutations altered the molecular weight of prephenate dehydratase. However, the intrinsic activity of prephenate dehydratase and its sensitivity to allosteric effectors was altered substantially following the initial step of partial purification.

Chorismate mutase of Chlamydomonas reinhardi: Partial purification and some properties

Chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5) was extracted from Chlamydomonas reinhardi by sonication. Fractionation of crude sonic extracts with (NH 4) 2 SO 4 and by DEAE-cellulose and Sephadex gel chromatography indicated a single peak of chorismate mutase activity with molecular weight 61 000. The Michaelis constant for 20-fold purified enzyme was 0.46 mM. Prephanate dehydrogenase (EC 1.3.1.9) and prephenate dehydratase (EC 4.2.1.40) activities were not detected in our crude or partially purified preparations of chorismate mutase. Tyrosine (1.25 mM) inhibited chorismate mutase activity by approx. 85% in crude and partially purified preparations. Phenylalanine (1.25 mM) inhibited 20%. Tryptophan (1.25 mM) by itself had no detectable effect on chorismate mutase activity but it completely reversed inhibition by tyrosine and phenylalanine. No repression of chorismate mutase was observed when the minimal growth medium was supplemented with automatic end products.

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16. II. The isolation and characterization of mutants auxotrophic for phenylalanine and tyrosine.

1. Mutants derived from the hydrogen bacterium Alcaligenes eutrophus strain H 16 auxotrophic for phenylalanine and tyrosine were isolated employing mutagenic agents (EMS, nitrite), the colistine counterselection technique and the "pin-point" isolation method. Three different types of mutants were found: (1) Mutants, requiring phenylalanine or phenylpyruvate for growth, were affected in chorismate mutase as well as prephenate dehydratase. Both activities were regained by reversion to prototrophy. The auxotrophic strains accumulated chorismic acid. (2) Strains with a growth response similar to that of the first group lacked only prephenate dehydratase activity which was partially regained by reversion. Chorismate mutase and prephenate dehydrogenase were derepressed up to two-fold. Mutants grown in minimal medium excreted prephenic acid. (3) The third type of mutants required phenylalanine or phenylpyruvate and grew slowly when supplemented with chorismate or prephenate. The enzymes involved in the specific pathway of phenylalanine and tyrosine were found to be present. Some of them were even more active than in the wild-type. 2. Mutants accumulating chorismic acid or prepheric acid were able to grow on minimal medium when incubated long enough. The chemical instability of the excretion products resulted in their nonenzymatic conversion to subsequent intermediates which were taken up by the cells, allowing growth. 3. A method is described for preparing barium prephenate using the auxotrophic mutant 6B-1 derived from A.eutrophus H 16. Prephenic acid, excreted by this strain, was obtained from the culture filtrate with a purity of at least 70% and a yield of approximately 180 mg per 21 of medium.

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase fromCorynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10−2mm and 10−1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.

Production of aromatic amino acids by microorganisms. VI. Regulatory properties of prephenate dehydrogenase and prephenate dehydratase from Corynebacterium glutamicum.

Regulatory properties of the enzymes in L-tyrosine and L-phenylalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by L-tyrosine. Prephenate dehydratase was strongly inhibited by L-phenylalanine and L-tryptophan and 100% inhibition was attained at the concentrations of 5×10-2mM and 10-1mM, respectively. L-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1mM) and restored the enzyme activity inhibited by L-phenylalanine or L-tryptophan. These regulations seem to give the balanced synthesis of L-tyrosine and L-phenylalanine. Prephenate dehydratase from C. glutamicum was stimulated by L-methionine and L-leucine similarly to the enzyme in Bacillus subtilis and moreover by L-isoleucine and L-histidine. C. glutamicum mutant No. 66, an L-phenylalanine producer resistant to p-fluorophenylalanine, had a prephenate dehydratase completely resistant to the inhibition by L-phenylalanine and L-tryptophan.

Cellular compartmentation of aromatic amino acids in Neurospora crassa. II. Synthesis and misplaced accumulation of phenylalanine in phen-2 auxotrophs.

Two mutants which require phenylalanine for normal growth and which show no prephenate dehydratase activity in vitro have been found to accumulate and excrete phenylalanine when incubated on minimal medium or grown on low concentrations of phenylalanine. The high levels of phenylalanine accumulated in these mutants apparently cannot be used for protein synthesis or for the regulation of the biosynthetic enzymes in the aromatic pathway. Mutant mycelia grown in high phenylalanine maintain a much lower level of free phenylalanine in the cells than do those grown on low phenylalanine or those which eventually grow on minimal. If all the phenylalanine required for the protein in a 3-day mycelial pad is supplied, little or no phenylalanine can be found in the medium after 3 days: if only a fraction of the total protein phenylalanine is supplied, the concentration of phenylalanine in the medium after 3 days is actually higher than the initial concentration. It is proposed that the mutation in these organisms has resulted in abnormal compartmentation of the phenylalanine produced so it cannot be utilized by the cells until it has been excreted and transported back into the normal pool channels. In this case, the transport (exogenous) and protein synthesis pools would be involved. The abnormal mislocation of the phenylalanine in the cell might be a result of the diffusion of free prephenate to low pH regions in the cell where it is nonenzymatically converted to phenylpyruvate. If, however, the mutant prephenate dehydratase is active in vivo, the mutation must somehow affect the activity or stability of the enzyme in vitro and also cause the release of the end product in the wrong place in the cell. This might be expected if the normal wild-type prephenate dehydratase is directionally oriented, e.g., as a result of membrane association, to release the product into normal cell channels (protein synthesis pool) while such oriented release might not occur in the mutants.

Regulation of phenylalanine biosynthesis in Pseudomonas aeruginosa

1. 1. The regulation of phenylalanine biosynthesis in Psuedomonas aeruginosa was examined. The enzymes studies were chorismate mutase, prephenate dehydratase and phenylalanine transaminase. 2. 2. Neither phenylalanine nor tyrosine affected the synthesis of chorismate mutase, prephenate dehydratase or phenylalanine transaminase. 3. 3. Chorismate mutase activity was inhibited by both phenylalanine and tyrosine. Prephenate dehydratase activity was inhibited by phenylalanine and stimulated by tyrosine. 4. 4. Chromatography on DEAE-cellulose showed only one major peak of activity for each of the three enzymes examined. 5. 5. The results are discussed in relation to the regulation of phenylalanine synthesis and to the occurrence of multi enzyme activities in other organisms.

Enzymatic characterization of a phenylalanine auxotroph of the blue-green bacterium Synechococcus cedrorum.

A phenylalanine-requiring strain of the unicellular blue-green bacterium Synechococcus cedrorum was isolated by means of penicillin enrichment following mutagenesis with nitrosoguanidine. Assays of pertinent enzymes indicated that prephenate dehydratase activity was absent in the mutant.

Channel-Shuttle Mechanism for the Regulation of Phenylalanine and Tyrosine Synthesis at a Metabolic Branch Point in Pseudomonas aeruginosa

A bifunctional protein complex was partially purified from Pseudomonas aeruginosa. Catalytic activities for chorismate mutase and prephenate dehydratase coeluted from gel filtration and DEAE-cellulose chromatography columns. The protein complex had a molecular weight of approximately 134,000, as determined by gel filtration. In crude extracts or in partially purified preparations about one-half of the chorismate utilized by the complex is converted to phenylpyruvate, and the other half accumulates as prephenate. The chorismate mutase activity is strongly product-inhibited by prephenate, competitively with chorismate. Accordingly, the first reaction of the complex can be sufficiently retarded by prephenate so that all of the reaction product is phenylpyruvate. Chorismate mutase activity is also competitively inhibited by phenylalanine. Although phenylalanine is effective at low concentrations, maximal inhibition is only 50 to 60%. Inhibition of chorismate mutase by phenylalanine was completely lost after gel filtration. The prephenate dehydratase activity of the protein complex is nearly completely inhibited by 0.1 mm phenylalanine in either crude extracts or partially purified preparations. A second species of prephenate dehydratase was separated from the prephenate dehydratase-chorismate mutase aggregate by gel filtration or anion exchange chromatography. The second prephenate dehydratase had an estimated molecular weight of 76,000, a high affinity for prephenate, and was insensitive to feedback inhibition by phenylalanine. The physiological role of the latter enzyme is uncertain. The other regulatory enzymes of tyrosine and phenylalanine biosynthesis, prephenate mutase aggregate by gel filtration or anion exchange chromatography. The other regulatory enzymes of tyrosine and phenylalanine biosynthesis, prephenate dehydrogenase (molecular weight of 120,000) and 3-deoxy-d-arabino-heptulosonate-7-phosphate synthetase (molecular weight of 52,000), elute from Sephadex G-100 columns as fractions which are distinct from both the chorismate mutase-prephenate dehydratase complex and from the low-molecular-weight species of prephenate dehydratase. A shuttle mechanism governing the metabolic fate of prephenate (to phenylalanine or to tyrosine) is proposed in the context of a model which also accommodates several previously puzzling findings: (i) the dominating role of tyrosine in the control of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthetase and (ii) the lack of feedback control of prephenate dehydrogenase by tyrosine.

Regulation of chloramphenicol synthesis in Streptomyces sp. 3022a. Branch-point enzymes of the shikimic acid pathway.

The pattern of induction and regulatory properties of the enzymes chorismate mutase, prephenate dehydratase, and anthranilate synthetase were investigated in a chloramphenicol-producing Streptomycete under a variety of environmental conditions. The enzymes were not subject to repression by intermediates or end products of the shikimic acid pathway. Throughout purification procedures chorismate mutase and prephenate dehydratase activities were each represented by single enzyme proteins of molecular weights 75 000 and 220 000, respectively. Chorismate mutase activity was not inhibited by end products or intermediates of the shikimic acid pathway. Prephenate dehydratase activity was controlled solely by feedback inhibition by L-phenylalanine, and anthranilate synthetase activity was only inhibited by L-tryptophan.

Repression of aromatic amino acid biosynthesis in Escherichia coli K-12.

Mutants of Escherichia coli K-12 were isolated in which the synthesis of the following, normally repressible enzymes of aromatic biosynthesis was constitutive: 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetases (phe and tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A. In the wild type, DAHP synthetase (phe) was multivalently repressed by phenylalanine plus tryptophan, whereas DAHP synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A were repressed by tyrosine. DAHP synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase were also repressed by phenylalanine in high concentration (10(-3)m). Besides the constitutive synthesis of DAHP synthetase (phe), the mutants had the same phenotype as strains mutated in the tyrosine regulatory gene tyrR. The mutations causing this phenotype were cotransducible with trpA, trpE, cysB, and pyrF and mapped in the same region as tyrR at approximately 26 min on the chromosome. It is concluded that these mutations may be alleles of the tyrR gene and that synthesis of the enzymes listed above is controlled by this gene. Chorismate mutase P and prephenate dehydratase activities which are carried on a single protein were repressed by phenylalanine alone and were not controlled by tyrR. Formation of this protein is presumed to be controlled by a separate, unknown regulator gene. The heat-stable phenylalanine transaminase and two enzymes of the common aromatic pathway, 5-dehydroquinate synthetase and 5-dehydroquinase, were not repressible under the conditions studied and were not affected by tyrR. DAHP synthetase (trp) and tryptophan synthetase were repressed by tryptophan and have previously been shown to be under the control of the trpR regulatory gene. These enzymes also were unaffected by tyrR.

Metabolic Interlock: THE MULTI-METABOLITE CONTROL OF PREPHENATE DEHYDRATASE ACTIVITY IN BACILLUS SUBTILIS

Abstract Regulatory interactions which relate small molecules and enzymes that are located in different sequences of biochemical reactions have been termed metabolic interlock. One of the simplest examples of the phenomenon occurs in the branching biochemical pathway for the synthesis of aromatic amino acids in Bacillus subtilis. Although prephenate dehydratase is not a member enzyme of the tryptophan-specific biosynthetic pathway, tryptophan inhibits the activity of prephenate dehydratase as effectively as the feedback inhibition exerted by phenylalanine. The inhibitions by tryptophan and phenylalanine are competitive with respect to prephenic acid, the substrate for the enzyme. Tyrosine, the end product of the third branch of the pathway, reverses the inhibition of prephenate dehydratase by tryptophan although it does not exhibit a strong allosteric effect alone. Several experiments showed that the allosteric specificities of prephenate dehydratase for tryptophan and tyrosine function in vivo. It was possible to show physiological conditions under which wild type cells were susceptible to growth inhibition by tryptophan. These conditions were equated with lower levels of intracellular tyrosine. More striking effects were noted by using mutant strains having a partial requirement for tyrosine. Hence, a mechanism appears to operate which decreases intracellular phenylalanine levels whenever intracellular levels of tyrosine are limiting or depleted. Methionine and leucine, allosteric activators of prephenate dehydratase, were found to reverse growth inhibition caused by tryptophan. The enzyme properties of several classes of mutants selected for resistance to growth inhibition by tryptophan are described.