/. Biochem., 76, 1103-1111 (1974)

Regulation of Prephenate Dehydratase in Brevibacterium flavum Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021

Shinichi SUGIMOTO and Isamu SHIIO Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki, Kanagawa 244

Received for publication, March 28, 1974

The regulation and some other properties of prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] of Brevibacterium flavum were studied. Prephenate dehydratase was purified about 38-fold from crude extracts of a tyrosine auxotroph, BX-1. The optimum pH of the reaction was 6.5 in potassium phosphate • buffer. The relationship between the enzyme concentration and activity was linear in the presence of tyrosine but not in its absence. Km for prephenate was 54 pM.. The enzyme was completely inhibited by phenylalanine or by tryptophan, while tyrosine not only reversed the inhibition competitively but also stimulated the enzyme activity about 10-fold. The concentration of tyrosine giving half-maximum activation was 1.6 ^M. Tyrosine affected both the Km value and the maximum reaction velocity. Similarly, phenylalanine and tryptophan were mixed-type inhibitors. The concentration of tryptophan giving 50% inhibition was 25 //M, 10 times that of phenylalanine, 2.5 fiM. None of the tyrosine and phenylalanine analogues tested strongly activated the enzyme, but several analogues inhibited it, as did phenylalanine. The molecular weight which was estimated to be 2.2 xlO5 by gel filtration experi- ments ; this value was not affected by tyrosine or by phenylalanine. On the basis of these results, the regulatory mechanism of aromatic amino acid biosynthesis in B. flavum was discussed.

Since some mutants of Brevibacterium flavum by tryptophan, while 3-deoxy-D-arafo>z0-hep- accumulate large amounts of tryptophan (/), tulosonate 7-phosphate synthetase (DAHP syn- phenylalanine, and tyrosine (2), it is of in- thetase*), the first enzyme in the common terest to study the regulatory mechanism of pathway, is synergistically inhibited by phenyl- aromatic amino acid biosynthesis in these or- alanine plus tyrosine and is repressed by tyro- ganisms. It has previously been reported that sine in B. flavum {3, 4). anthranilate synthetase [EC 4.1. 3.27], the first Prephenate dehydratase [prephenate hydro- enzyme in the tryptophan-specific biosynthetic lyase (decarboxylating), EC 4.2.1.51], the first pathway, is strongly inhibited and repressed enzyme in the phenylalanine-specific pathway, has been reported to be regulated by strong * The following abbreviation is used: 3-deoxy-D- end-product (phenylalanine) inhibition in vari- araWwo-heptulosonate 7-phosphate: DAHP. ous bacteria (5—7). However, mutants derived

Vol. 76, No. 5, 1974 1103 1104 S. SUGIMOTO and I. SHIIO from B. flavum No. 2247, in which DAHP also contained 500 mg/liter tyrosine for the synthetase had been genetically desensitized to tyrosine auxotroph, BX-1. feedback inhibition by phenylalanine plus tyro- Chemicals—Barium prephenate was pre- sine, over-produced large amounts of phenyl- pared from chorismic acid, as described pre- alanine and tyrosine (2). This result cannot viously (2). Amino acids used were all In- be explained simply by the feedback inhibition form unless otherwise cited and were purchased of prephenate dehydratase by phenylalanine. from Sigma, Calbiochem or Nutritional Bio-

A metabolic interlock on prephenate de- chemicals Corp. D-Phenylalanine and D-tyro- Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021 hydratase has been observed in some bacteria. sine were the products of Nutritional Biochem- For example, prephenate dehydratase of Bacil- icals Corp. and o-hydroxy-DL-phenylalanine, lus subtilis is activated by leucine and methio- m - hydroxy - DL - phenylalanine, p - amino - DL - nine, but inhibited by tryptophan. Further- phenylalanine, w-fluoro-DL-phenylalanine, p- more tyrosine reverses the inhibition by trypto- fluoro - DL - phenylalanine, p - hydroxyphenyl - phan but not that by phenylalanine (8, 9). pyruvic acid, />-aminobenzoic acid, and p- On the other hand, prephenate dehydratase of hydroxybenzoic acid were from Sigma. Mark- Pseudomonas sp. is activated by tyrosine (7). ers used in the gel filtration experiments were These findings also fail to explain the accumu- glutamate dehydrogenase [EC 1.4.1.3] from lation of both tyrosine and phenylalanine as bovine liver, molecular weight 300,000 (18) described above, even if such interactions exist (Nutritional Biochemicals Corp.), catalase [EC in B. flavum. 1.11.1.6] from bovine liver, molecular weight Preliminary experiments with crude ex- 244,000 (79) (Sigma), alcohol dehydrogenase tract of B. flavum showed that prephenate de- [EC 1.1.1.1] from yeast, molecular weight hydrogenase [EC 1.3.1.12], the first enzyme 150,000 (20) (Boehringer), hexokinase [EC in the tyrosine-specific pathway, is not affected 2.7.1.1] from yeast, molecular weight 96,600 by the end-product, tyrosine, while prephenate (21) (Boehringer), and cytochrome cfrom horse dehydratase is not only inhibited by phenyl- heart, molecular weight 12,400 (22) (Tokyo alanine, but also activated by tyrosine (2). Kasei). Furthermore, the phenylalanine inhibition is Enzyme Assay — Prephenate dehydratase reversed by tyrosine. Since these effects of activity was measured by the spectrophoto- tyrosine on prephenate dehydratase could well metric determination of phenylpyruvate formed explain the phenomenon of tyrosine and phenyl- in the reaction according to Cotton and Gibson alanine accumulation by mutants, prephenate (5). The standard assay system contained 20 dehydratase was further purified from B. flavum //moles of Tris-HCl buffer, pH 7.5, 0.2 //mole and its regulatory and some other properties of barium prephenate, 0.2 //mole of EDTA and were studied, as reported in this paper. enzyme preparation in a total volume of 0.4 ml. The reaction was carried out at 30° for MATERIALS AND METHODS 20 min and stopped by the addition of 0.8 ml of 1N NaOH. The absorbance of phenyl- Organisms and Cultivation—Brevibacterium pyruvate formed in the reaction was measured flavum No. 2247 (ATCC 14067) and its tyrosine at 320 nm. One unit of the enzyme activity auxotroph, BX-1 (1) were aerobically cultured was defined as the amount catalyzing the for- for 24 hr at 30° in a glucose medium (36 g of mation of 1 nmole of phenylpyruvate per min, glucose, 10 g of urea, 1 g of KH2PO4, 0.4 g of taking the molar extinction coefficient as 17,500 MgSO«-7H2O, 10 mg of FeSO4-7H2O, 2 ppm at 320 nm. 2+ Mn , 100 /*g of thiamine-HCl, 1 ml of Mieki,* Protein was determined by the method of 30 fig of d-biotin, 7 ml of 6 N HC1, and dis- Lowry et al. (10). tilled water to a total volume of 1 liter) which Preparation of the Enzyme—After cultiva- tion of the tyrosine auxotroph, BX-1 in the * Mieki is a hydrolysate of soybean meal, a product glucose medium, the cells were harvested, of Ajinomoto Co., Inc. washed twice with 0.2% KC1, suspended in

/. Biochem. REGULATION OF PREPHENATE DEHYDRATASE 1105

0.1 M Tris-HCl buffer, pH 7.5 and sonically sulfate. The precipitate was dissolved in a disrupted at 10 kc for 20 min. Crude extract small volume of the same buffer (Fraction V) (Fraction I) obtained by centrifugation of the and used throughout this study unless other- sonicate at 32,000 xg for 30 min at 0° was wise stated. treated with solid ammonium sulfate. The precipitate between 0.25 and 0.60 saturation of RESULTS ammonium sulfate was collected by centrifuga- tion and was dissolved in 0.1 M Tris-HCl buffer, Partial Purification and General Properties pH 7.5 (Fraction II). Fraction II was then of Prephenate Dehydratase from the Tyrosine Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021 treated with saturated ammonium sulfate so- Auxotroph, BX-1—Since, the substrate of pre- lution and the precipitate between 0.38 and phenate dehydratase, i.e., prephenate, is com- 0.50 saturation of ammonium sulfate was dis- mon to the prephenate dehydrogenase step in solved in a small volume of 0.05 M Tris-HCl the tyrosine-specific pathway, the prephenate buffer, pH 7.5 (Fraction III). Fraction III was dehydratase reaction would be affected by pre- dialyzed against about 200 volumes of the same phenate dehydrogenase, if it was present in buffer at 5° for 16 hr and was then applied to the enzyme preparation. Therefore, prephenate a DEAE-cellulose column (2.5x23 cm) equili- dehydratase was purified from the crude ex- brated with the same buffer. The column tract of the tyrosine auxotroph, BX-1, which was washed with one volume of the same genetically lacked prephenate dehydrogenase. buffer and then linear gradient elution was The results on the partial purification are shown started with the same buffer containing 0.5 M in Table I. Prephenate dehydratase was puri- KC1 at a flow rate of 25 ml/hr. Eight-ml frac- fied about 38-fold from crude extract as de- tions were collected. The fractions containing scribed in "METHODS." The total activity of prephenate dehydratase were pooled and treated Fraction II was higher than that of Fraction with ammonium sulfate to give 0.70 saturation. I, probably due to removal of the inhibitor, The precipitate was dissolved in a small vol- phenylalanine, as mentioned later. Activities ume of the same buffer (Fraction IV). Frac- of DAHP synthetase, chorismate mutase [EC tion IV was passed through a Sephadex G-200 5.4. 99.5], and prephenate dehydrogenase were column (2.5x45 cm) equilibrated with the same not observed at all in the Fraction V prepa- buffer at a flow rate of 10 ml/hr. Two-ml ration, which was used as the enzyme prepa- fractions were collected. The fractions con- ration throughout this study. taining prephenate dehydratase were collected The optimum pH of the reaction was 6.5 and brought to 0.70 saturation with ammonium in potassium phosphate buffer. However, Tris-

TABLE I. Purification of prephenate dehydratase. Tyrosine auxotroph of B. flavum, strain BX-1, was cultured in glucose medium containing 500 mg/liter of L-tyrosine at 30° for 24 hr. Methods for the isolation and purification of the enzyme are described in "METHODS." Prephenate dehydratase activities were determined in the standard assay medium supplemented with 1 mM tyrosine.

Total protein Total activity Specific activity Step and fraction (mg) (unitt) (unit/mg protein)

I. Crude extract 2,180 20,700 9.5 II. Ammonium sulfate (0.25-0.60 saturation) 1,600 25,000 16 III- Ammonium sulfate (0.38-0.50 saturation) 308 14,600 48 IV. DEAE-cellulose 22 4,760 217 V. Sephadex G-200 4.2 1,530 360

t One unit of activity is equivalent to the formation of 1 nmole of phenylpyruvate per min.

Vol. 76, No. 5, 1974 1106 S. SUGIMOTO and I. SHIIO HC1 buffer pH 7.5, was used in the standard Double reciprocal plots of the reaction rate assay medium for comparison with other re- against substrate concentrations were almost lated enzymes under the same conditions and linear, as shown in Fig. 4 and Fig. 6 when for studying metabolic regulation in aromatic the concentrations are relatively high. The .amino acid biosynthesis. Km value for prephenate was calculated to be As shown in Fig. 1, the relationship be- 54 [xM. tween the enzyme activity and the concentra- Effects of Aromatic Amino Acids on Pre- tion of the enzyme was completely linear in phenate Dehydratase—-The effects of aromatic the presence of 1 mM tyrosine, which was an amino acids were examined at 0.1 mM, as Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021 activator. However, the plots bend upward shown in Table II. The enzyme was inhibited in the absence of tyrosine. This suggests a by phenylalanine as well as by tryptophan, certain transition in the molecular state de- but was strongly activated by tyrosine. Fur- pending on the concentration of the enzyme. thermore, equimolar mixtures of tyrosine plus The molecular weight of prephenate de- phenylalanine, tyrosine plus tryptophan or hydratase was determined by gel filtration ex- tyrosine, phenylalanine and tryptophan, all ac- periments on a Sephadex G-200 column. From tivated the enzyme. That is, tyrosine released logarithmic plots of the peak positions of the the enzyme activity from inhibition by phenyl- markers against their molecular weights, the alanine and/or tryptophan almost completely. molecular weight of prephenate dehydratase Effects of Tyrosine and Phenylalanine was estimated to be about 2.2 xlO5 by the Analogues on Prephenate Dehydratase—To ex- method of Andrews (11), as shown in Fig. 2. The molecular weight of prephenate dehydra- tase was not altered in the presence of tyrosine and phenylalanine and remained the same at enzyme concentrations between 3.8 units/ml and 145 units/ml. This suggests that the change of the enzyme molecular state suggested from Fig. 1 may not involve alteration of the molecular weight.

70 90 110 130 150 ELUTION VOLUME (ml) Fig. 2. Determination of the molecular weight of prephenate dehydratase by gel filtration on Sephadex G-200. The column (1.8x61 cm) was equilibrated with 0.05 M Tris-HCl buffer, pH 7.5. Two milli- grams of prephenate dehydratase and markers (1 mg of glutamate dehydrogenase, 0.3 mg of catalase, 0.06 mg of alcohol dehydrogenase, 2 mg of hexo- 20 40 60 80 kinase and 3 mg of cytochrome c) were combined, ENZYME (unit/ml) and simultaneously passed through the column. Fig. 1. Effects of prephenate dehydratase concen- Elution was carried out at 5° at a flow rate of 10 tration on the reaction velocity in the presence and ml/hr. Abbreviations: glutamate dehydrogenase from absence of tyrosine. The reaction was carried out bovine liver (A), catalase from bovine liver (B), pre- under the standard assay conditions. At the highest phenate dehydratase from B. flavum (C), alcohol de- enzyme concentration, 25 //g of protein (Fraction V) hydrogenase from yeast (D), hexokinase from yeast was used. O, no tyrosine; •, 1 mM tyrosine. (E), and cytochrome c from horse heart (F).

/. Biochem. REGULATION OF PREPHENATE DEHYDRATASE 1107

amine the specificity of activation by tyrosine DL - phenylalanine and p - fiuoro - DL - phenyl - and of inhibition by phenylalanine, the effects alanine) also inhibited the enzyme. />-Hydroxy- of tyrosine and phenylalanine analogues were phenylpyruvate, which is a precursor in tyro- tested, as shown in Table II. None of the sine biosynthesis, />-amino-DL-phenylalanine, p- analogues tested activated prephenate dehydra- aminobenzoic acid, and />-hydroxybenzoic acid tase except D-tyrosine, which increased the all had no significant effect on the enzyme activity only about 1.5-fold. Analogues with activity. Therefore, it seems that the activa- an ortho or meta hydroxy group instead of the

tion effect of tyrosine in much more struc- Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021 para hydroxy group in tyrosine, i.e., o-hydroxy- turally specific than the inhibitory effect of DL-phenylalanine and ra-hydroxy-DL-phenyl- phenylalanine. alanine, inhibited the enzyme activity, as did Whereas Jensen has reported activation phenylalanine. Analogues with m-fluoro and by methionine and leucine of prephenate de- £-fluoro substituents of phenylalanine (w-fluoro- hydratase in Bacillus subtilis (8), methionine and leucine at 10 mM had no effect on pre- TABLE II. Effects of aromatic amino acids, tyrosine phenate dehydratase in B. flavunt. and phenylalanine analogues, aromatic vitamins and Activation by Tyrosine—Figure 3 shows the other amino acids on prephenate dehydratase. The effects of tyrosine concentration on prephenate activities were measured in the presence and absence dehydratase. The enzyme was strongly acti- of given addition(s) under the standard assay con- vated by tyrosine; the concentration giving ditions and were expressed as percentage of the half-maximum activation was 1.6 fiM. The control. Fraction V enzyme, 22 fig of protein, was activity reached a maximum on the addition used, except when the relative activity was more of 50 fiM tyrosine, about 10 times as high as than 400%, in which case 4.5 fig of protein was used. that in the absence of tyrosine. However, Concen- Relative since the relationship between the activity and Addition tration activity the concentration of the enzyme was linear (mM) (%) only in the presence of tyrosine, as indicated None (control) — 100 in Fig. 1, the maximum activation depended on the enzyme concentration. Tyrosine 0.1 630 Double reciprocal plots of the reaction rate Phenylalanine 0.1 8 in the presence and absence of tyrosine are Tryptophan 0.1 11 shown in Fig. 4. The plots were almost linear Tyrosine + phenylalanine 0.1 525 in the presence and absence of tyrosine at Phenylalanine + tryptophan 0.1 5 substrate concentrations higher than 25 fiM, Tryptophan + tyrosine 0.1 622 Tyrosine+phenylalanine +tryptophan 0.1 483- D-Tyrosine 1.0 147 D-Phenylalanine 1.0 13 o- Hydroxy- DL -phenylalanine 1.0 13 ;n-Hydroxy-DL- phenylalanine 1.0 7 wt-Fluoro-DL-phenylalanine 1.0 4 />-Fluoro-DL- phenylalanine 1.0 5 p-Amino-DL- phenylalanine 1.0 104 />-Hydroxyphenylpyruvic acid 0.1 108 7 6 5 4 3 />-Aminobenzoic acid 1.0 106 TYROSINE CONCENTRATION (logOO) />-Hydroxybenzoic acid 1.0 109 Fig. 3. Effect of tyrosine concentration on prephenate L-Methionine 10 104 dehydratase. Fraction V enzyme, 4.7 fig of protein, L-Leucine 10 109 was used under the standard assay conditions. Tyrosine was added the concentrations indicated.

Vol. 76, No. 5, 1974 1108 S. SUGIMOTO and I. SHIIO

TABLE III. Prephenate dehydratase activities in cell-free extracts of B. flavum No. 2247 and the tyrosine auxotroph, BX-1. The reaction was carried out under the standard assay conditions with or without the addition of 1 HIM tyrosine. The cell- free extracts (about 250 f/g) or their gel nitrates (about 250 fig) through a Sephadex G-50 column (1.5x10 cm) equilibrated with 0.05 M Tris-HCl buffer,

pH 7.5, were used as the enzyme. Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021

Tyrosine Specific added activity Strain Sample / unit/mg \ (lmM) \ protein /

No. 2247 Cell-free extract — 3.3 (wild) Cell-free extract 4- 7.9 0 10 40 80 1/(PREPHENATE) (HIM) Gel filtrate - 0.91 Gel filtrate + 7.1 Fig. 4. Double reciprocal plots of reaction rate against prephenate concentration in the presence BX-1 Cell-free extract — 0.80 and absence of tyrosine. Fraction V enzyme, 8.0 (Tyr") Cell-free extract + 6.5 fig of protein, was used under the standard assay conditions except for the substrate concentrations. Gel filtrate - 1.4 Tyrosine was added at 0 (•), 0.5 (A), or 1 ftM (O). Gel filtrate + 9.0 and indicated that activation by tyrosine affect- tyrosine seems not to be due to inactivation ed both the Km value and the maximum re- of the enzyme but rather to removal of a action velocity. At lower concentrations, the certain activator which may exist in the cell- plots bend upward. free extract. The specific activity of the ex- Possible Operation of Tyrosine Activation tract from tyrosine auxotroph, BX-1 was 0.80 in Vivo—As mentioned above, the half-maxi- without tyrosine, much lower than that of the mum concentration of tyrosine for activation wild strain, whereas in the presence of tyro- is extremely low, 1.6 pM, which is much lower sine, the specific activity was almost the same than the phenylalanine and tyrosine concen- as that of the wild strain. Moreover, the tration giving 50% inhibition of DAHP synthe- specific activity without tyrosine did not de- tase, 13 ^M (4). Since tyrosine concentration crease on gel filtration, in contrast to that of in the cell seems to be controlled through the the wild strain. These results suggest that feedback inhibition of DAHP synthetase, which the tyrosine auxotroph, BX-1 is unable to is the only significant control mechanism in produce the activator which is produced by the tyrosine biosynthetic pathway, the data the wild strain. Since this strain lacks pre- suggest active operation of tyrosine control phenate dehydrogenase and therefore cannot effects in vivo. Table III shows the prephenate synthesize tyrosine or £-hydroxyphenylpyru- dehydratase activities of cell-free extracts from vate (of which the former alone activates pre- B. flavum wild strain No. 2247 and the tyro- phenate dehydratase), it is likely that the ac- sine auxotroph, BX-1. The specific activity of tivator may be tyrosine. Thus, it appears that the cell-free extract from the wild strain was prephenate dehydratase might have been par- 3.3 without the addition of tyrosine and de- tially activated by tyrosine formed in vivo in creased to 0.91 after gel filtration on a Sepha- the wild strain. dex G-50 column. Since the activities of both Inhibition by Phenylalanine—As shown in the cell-free extract and the filtrate were al- Fig. 5, prephenate dehydratase activity was most the same when tyrosine was added, the completely inhibited by phenylalanine, and decrease of the activity in the absence of phenylalanine inhibition was strongly affected

/. Biochem. REGULATION OF PREPHENATE DEHYDRATASE 1109

100 Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021

-7 -6 -5 -4" -3 -2 PHENYLALANINE CONCENTRATION (logM) 0.1 0.5 Fig. 5. Effects of phenylalanine concentration on l/CTYROSINE) prephenate dehydratase. The reaction was carried Fig. 7. Double reciprocal plots of reaction rate out under the standard assay conditions in the pres- against tyrosine concentration in the presence and ence and absence of tyrosine. The concentrations absence of phenylalanine. Fraction V enzyme, 8.0 of tyrosine and the amounts of enzyme were 0 and fig of protein, was used under the standard assay 27 fig (O), 2 fiu and 9.5 fig (A), 50 ftu and 8.0 fig conditions. Phenylalanine concentrations were0(0), (D), and 1 DIM and 4.7 fig (•), respectively. 2 fiM (A), 5 fiM (D), or 10 fiM (•).

100

-7 -6 -5 -4 ~^3 0 5 20 40 TRYPTOPHAN CONCENTRATION (logCM]) 1/CPREPHENATE) (CUM) Fig. 8. Effects of tryptophan concentration on pre- Fig. 6. Double reciprocal plots of reaction rate phenate dehydratase. The reaction was carried out against prephenate concentration in the presence under the standard assay conditions in the presence and absence of phenylalanine. Fraction V enzyme, or absence of tyrosine. The concentrations of tyro- 12 fig of protein, was used under the standard assay sine and the amounts of enzyme were 0 and 25 fig conditions except for the substrate concentrations. (O), 2 fiM and 13 fig (A), and 50 fiM and 8.0 fig Phenylalanine concentrations were 0 (O), 2 /ZM (A), (•), respectively. 3 ftM (•) or 4 ftu (•). that phenylalanine is a mixed-type inhibitor, by the simultaneous addition of tyrosine. with Ki and K,' values of 1.0 fiM and 2.7 ^M, Fifty % inhibition by phenylalanine took place respectively. at 2.5 pM, 6 ftM, 90 ftM, and 2.3 mM in the In Fig. 7, the relationship between tyro- presence of 0, 2 //M, 50 pM, and 1 mM tyro- sine and phenylalanine is shown by double sine, respectively. As shown in Fig. 6, double reciprocal plots of the reaction rate against reciprocal plots of the reaction rate in the ab- tyrosine concentrations in the presence and sence and presence of phenylalanine indicate absence of phenylalanine. The curves meet

Vol. 76, No. 5, 1974 1110 S. SUGIMOTO and I. SHIIO at one point on the ordinate, indicating that enzyme is inhibited by />-fluorophenylalanine tyrosine and phenylalanine affected the reaction but not by D-phenylalanine (12). competitively. It has been found that prephenate dehy- Inhibition by Tryptophan—As shown in dratase associates with chorismate mutase in Fig. 8, tryptophan completely inhibited the a bifunctional enzyme complex in Aerobacter enzyme activity, as did phenylalanine, and aerogenes (5), E. coli (5), Salmonella typhi- this inhibition was reversed by the simultane- murium (13), and Pseudomonas aeruginosa ous addition of tyrosine. Concentrations of (14), whereas there is no such stable aggre- Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021 tryptophan giving 50% inhibition were 25 fiU, gate form in Neurospora crassa (15), Saccharo- 50 fiM, and 1.2 mM in the presence of 0, 2 //M, myces cerevisiae (6), Psium sativum (16), and and 50 ^M tyrosine, respectively; these con- Bacillus subtilis (17). In this respect, it is of centrations were about 10 times higher than interest that chorismate mutase activity was those of phenylalanine. not detected at all in the partially purified pre- Double reciprocal plots in the presence phenate dehydratase preparation from B. and absence of tryptophan gave straight lines, flavum. indicating that tryptophan is also a mixed-type Prephenate dehydratase of B. flavum was inhibitor with Ki and K! values of 24 /iM and also completely inhibited by tryptophan, but 45 piM, respectively. These values were about the concentration of tryptophan giving 50% 20 times higher than those for phenylalanine. inhibition was 25 fiM, about 10 times higher than that of phenylalanine. As reported pre- DISCUSSION viously (3), tryptophan biosynthesis in B. flavum is controlled by the strong feedback The present results show that prephenate de- inhibition of anthranilate synthetase; the hydratase of the phenylalanine-specific biosyn- tryptophan concentration giving 50% inhibition thetic pathway is completely inhibited by low is 1.6 liM. Therefore, phenylalanine biosyn- concentrations of L-phenylalanine in B. flavum, thesis would not be disturbed by tryptophan, as in many other organisms. As described unless tryptophan was exogenously added in previously (4), DAHP synthetase of the com- excess. Considering the low specificity of mon pathway for aromatic amino acid bio- phenylalanine inhibition, as mentioned above, synthesis is also inhibited by phenylalanine in it is likely that tryptophan may act as a the presence of tyrosine in B. flavum. How- phenylalanine analogue. In fact, kinetic-type of ever, since the inhibition of prephenate de- tryptophan inhibition and the effect of tyrosine hydratase was much stronger than that of are very similar to phenylalanine inhibition. DAHP synthetase, phenylalanine biosynthesis Furthermore, prephenate dehydratase desen- in this organism seems to be controlled by the sitized to phenylalanine feedback inhibition by feedback inhibition of prephenate dehydratase. mutation is also insensitive to inhibition by This is in good accord with the fact that tryptophan (unpublished data). Therefore, mutants having prephenate dehydratase insen- tryptophan acts at the same regulatory site as sitive to feedback inhibition excrete phenyl- phenylalanine. Tryptophan has also been found alanine (2). to inhibit prephenate dehydratase activity in In B. flavum, phenylalanine inhibition of Bacillus subtilis (9). In this case, the degree prephenate dehydratase was not very specific, of tryptophan inhibition is almost the same so that many L-phenylalanine analogues tested, as that of phenylalanine inhibition but the including D - phenylalanine, o - hydroxy - DL - effect of tyrosine on the inhibition is quite phenylalanine, and />-fluoro-DL-phenylalanine different. Therefore, it appears that trypto- showed strong inhibitory effects, in contrast phan may operate at a different regulatory to the strict specificity for phenylalanine in- site from phenylalanine. hibition in the Pseudomonas enzyme (7). The In B. flavum, low concentrations of tyro- specificity in the Bacillus enzyme is inter- sine not only activate prephenate dehydratase mediate between these two enzymes, as the but also reverse the inhibition of phenylalanine

/. Biochem. REGULATION OF PREPHENATE DEHYDRATASE 1111 and of tryptophan. Furthermore, specificity for the tyrosine activation was strict. Among REFERENCES analogues tested, D-tyrosine alone activated the enzyme slightly. Tyrosine activation was 1. I. Shiio, H. Sato, and M. Nakagawa, Agr. Biol. so effective that the half-maximum concentra- Chem., 36, 2315 (1972). 2. S. Sugimoto, M. Nakagawa, T. Tsuchida, and tion of tyrosine for activation was much less I. Shiio, Agr. Biol. Chem., 37, 2327 (1973). than the concentration giving 50% inhibition 3. I. Shiio, R. Miyajima, and M. Nakagawa, /. Bio- of DAHP synthetase. This suggests active chem., 72, 1447 (1972). Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021 operation in vivo of tyrosine activation. In 4. I. Shiio, S. Sugimoto, and R. Miyajima, /. Bio- fact, prephenate dehydratase in the cell-free chem., 75, 987 (1974). extract from the wild strain had already been 5. R.G.H. Cotton and F. Gibson, Biochim. Biophys. activated by tyrosine formed in the cells. Ada, 100, 76 (1965). As described previously (2), B. flavum 6. F. Lingens, W. Goebel, and H. Uesseler, Bio- mutants having DAHP synthetase insensitive chem. Z., 346, 357 (1966). to feedback inhibition excrete not only tyrosine 7. P. Cerutti and G. Guroff, /. Biol. Chem., 240, 3034 (1965). but also phenylalanine, although they have 8. R.A. Jensen, /. Biol. Chem., 244, 2816 (1969). normal prephenate dehydratase which is sensi- 9. J.L. Rebello and R.A. Jensen, /. Biol. Chem., tive to feedback inhibition by phenylalanine. 245, 3738 (1970). The over-production of tyrosine can easily be 10. O.H. Lowry, NJ. Rosebrough, A.L. Farr, and explained in terms of the absence of control RJ. Randal, /. Biol. Chem., 193, 265 (1951). of biosynthesis in these mutants. However, 11. P. Andrews, Biochem. /., 96, 595 (1966). the over-production of phenylalanine is difficult 12. J.H. Coats and E.W. Nester, /. Biol. Chem., to understand unless reversion of the feedback 242, 4948 (1967). inhibition of prephenate dehydratase by tyro- 13. J. Dayan and D.B. Sprinson, /. Bacteriol., 108, sine occurs. Thus it is likely that the phy- 1174 (1971). siological significance of the effects of tyrosine 14. S.I. Ahmed and J.J.R. Campbell,/. Bacteriol., 115, on prephenate dehydratase may involve main- 205 (1973). taining a balanced synthesis of phenylalanine 15. A.A. El-Eryani, Cenetics, 62, 711 (1969). 16. R.G.H. Cotton and F. Gibson, Biochim. Biophys. and tyrosine. Activation of prephenate dehy- Ada, 156, 187 (1968). dratase by tyrosine has been reported in 17. J.H. Lorence and E.W. Nester, Biochemistry, 6, Pseudomonas sp. (7). However, the degree 1541 (1967). of activation and the specificity for tyrosine 18. J.-C. Patte, P. Truffa-Bachi, and G.N. Cohen, in Pseudomonas sp. were less than those in B. Biochim. Biophys. Ada, 128, 426 (1966). flavum. D-Tyrosine activated the enzyme sim- 19. T. Samejima and J.T. Yang, /. Biol. Chem., 238, ilarly to L-tyrosine and />-aminophenylalanine 3256 (1963). also activated the enzyme considerably. How- 20. J.E. Hayes and S.F. Velick, /. Biol. Chem., 207, ever, tyrosine does not activate the prephenate 225 (1954). dehydratase of Bacillus subtilis; it reverses the 21. M. Kunitz and M.R. McDonald, /. Gen. Physiol., tryptophan inhibition but not that of phenyl- 29, 393 (1946). alanine (9). 22. E. Margoliash, /. Biol. Chem., 237, 2161 (1962).

The authors are indebted to Drs. M. Takahashi and H. Okada of their laboratories for encouragement during this work.

Vol. 76, No. 5, 1974