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Inhibition of thienylalanine incorporation in Escherichia coli by 2-amino-3-phenylbutanoic acid
ARCHIVES
OF
BIOCHEMISTRY
Inhibition
AND
103, 175-180 (1963)
BIOPHYSICS
of Thienylalanine
Incorporation
2-Amino-3-Phenylbutanoic JEROME From the Department
EDELSOIS’
of Chemistry,
Received
of Southwestern March
co/i by
Acid
AKD DEAN
University
in Escherichia
F. KEELEY Louisiana,
Lafayette,
Louisiana
28, 1963
2-Amino&phenylbutanoic acid was prepared via a typical malonic ester synthesis and shown to be an effective phenylalanine antagonist for Leuconostocdextranicum but not for Escherichia coli. However, it does competitively prevent the toxicity due to thienylalanine by inhibiting the incorporation of thienylalanine in E. coli. EXPERIMENTAL
INTRODUCTION
BIOLOGICAL ASSAYS
Studies of the biological effects of various phenylalanine analogs upon the growth of Leuconostoc dextranicum have shown that the steric configurations of the antagonists have a profound influence on their activities. The specificities of many nonaromatic analogs have been ascribed to the planarity and size of the group attached to the P-carbon of the alanine side chain (14). Substituent,s on the p-carbon of phenylalanine, e.g., fi-phenylserine (5), have also produced phenylalanine antagonists, and the present investigation was initiated in order to determine the effect upon biological activity of different substituents on the P-carbon
of the
natural
amino
For assays with L. dextranicum 8086, tyrosine and phenylalanine were omitted from a previously described amino acid medium (7), the salts A concentration was increased fourfold; the medium was further supplemented with 0.02 pg. per ml. pantethine and 0.2 pg. per ml. calcium pantothenate. The assays with E. coli W employed a salts-glucose medium (8). Mechanical details of this procedure have been reported in detail (9), except that, for cell counts, the medium was modified by the addition of agar to a final concentration of 2%. In all assays the amino acid analogs were dissolved in sterile water and added to sterile assay tubes without being heated. The amount of growth was determined turbidimetrically with a Bausch and Lomb Spectronic 20 calorimeter at 525 rnp, using distilled water to set 100% transmittance.
acid.
Accordingly, 2 - amino-3-phenylbutanoic acid (APBA) was prepared and its biological activity was investigated using L. dextranicum 8086 and Escherichia coli W as the assay organisms. Although not toxic for E’. co&i,APBA inhibits growth of L. dextranicum and its toxicity is competitively reversed by phenylalanine. In addition the toxicity of thienylalanine for E. coli, which may be reversed by analogs such or p-1-naphthylalanine
Wallace
Ethyl 2-Acetamido-d-CarbethoxyS-Phenylbutanoate
as p-tolylalanine
(6), may also be
relieved by APBA. 1 Present address: bury, New Jersey.
CHEMICAL SYNTHESIS
Laboratories,
Cran-
To a solution of 2.3 g. of sodium reacted with 200 ml. anhydrous ethanol, 20 g. of ethyl acetamidomalonate were added, followed by the addition of 18.5 g. of (I-bromoethyl)benzene. The reaction mixture was heated to reflux for 4 hr., and the inorganic salts which formed were removed by filtration. After pouring the alcoholic filtrate over ice, 13.5 g. of solids precipitated and were recrystallized from ethanol-water, m.p. 12P125”C.
175
176
EDELSON
Anal. Calcd. N, 4.50.
for CnH23NOb:
AND
N, 4.36. Found:
d-Amino-S-Phenylbutanoic
Acid
A mixture of 10 g. of ethyl 2-acetamido-2carbethoxy-3-phenylbutanoate and 100 ml. of 6 N hydrochloric acid was heated under reflux for 16 hr., and the resulting solution was evaporated to dryness in racuo. After two recrystalliaations from 6 N hydrochloric acid, 1.8 g. of sample was obtained, m.p. 200°C. dec.; reported m.p. 198”C., via a different method of preparation (10). Anal. Calcd. for CloH13N02.HCl: N, 6.50. Found: N, 6.31.
RADIOCHEMICAL
ASSAYS
Bacterial Studies Escherichia coli cells were grown in 10 ml. of salts-glucose medium (8) supplemented with 0.2 pg. per ml. of thienylalanine-j%Ci” added aseptically without heating, unless some other concentration is specified in the Tables or Figures. This concentration is not inhibitory and maximum growth is attained in about 15 hr. at 37°C. Incubation for a minimum of 18 hr. assures good growth. For washing effects and time studies, larger volumes were prepared and lo-ml. aliquots were taken after incubation. The cells were kept uniformly suspended by rapid stirring. The weight of cells was determined by turbidity measurements using a Beckman Model DU spectrophotometer. This was calibrated at 575 m by dilution of a culture of E. coli with medium, an aliquot of which was dried and weighed on a tared Millipore filter. Following separation of the suspended bacteria on a 47 mm. Millipore filter, type HA, the bacteria were subjected to three successive distilled water washes of 10, 10, and 80 ml. The samples were dried in an oven at 5960°C. and stored in a desiccator prior to counting. It was found that the filters could be conveniently held flat in a-inch stainless steel planchets during the drying and counting operations by means of g-inch segments of 1% inch iron pipe. The activity of the dried samples was measured by means of a Tracerlab Model FD-2 thin window (< 125 pg. per cm.2) gas flow counter employing methane-argon counting gas.
Determination
of the Detection
Coeficient
Ten PC. of a sample of P-2-thienyl-nn-alanine@-Cl4 (California Corporation for Biochemical Research) with a specific activity of 3.0 PC. per wmole was dissolved in 5.6 ml. of distilled water. The resulting solution, containing 100 pg. per ml. of the thienylalanine, was used as a stock solution for the entire study.
KEELEY
Twenty pl. of the thienylalanine stock solution was diluted to a volume of 50 ml. with distilled water. Four l.O-ml. aliquots of the diluted stock solution were evaporated to dryness in l-inch stainless steel planchets by means of a sample spinner-heat lamp assembly. The average activity, Ai, of the four samples was determined. Two loml. aliquots of the diluted stock solution were each treated with a sample of Norit weighing approximately 2 mg. The Norit was removed by filtration through a 47-mm., type HA Millipore filter, and dried without washing. The activity, A, of each sample was then determined. Four l.O-ml. aliquots of the filtrate from each of the Norit treated solutions were evaporated to dryness in 1 inch planchets and the average activity, A,, determined as before. The difference in average counting rates between the 1 ml. aliquot samples taken before and after Norit treatment was used to calculate the fraction, f, of the activity absorbed by the charcoal:
f=- Ai - A,
A< - Aa
where
Ai = initial activity of samples, cpm. Al = final activity of samples, cpm. & = From activity alanine sample k, was
background activity, cpm. the concentration of the solution, specific of thienylalanine, and fraction of thienylremoved, the activity, S, in each Norit was computed. The detection coefficient, then determined from the relationship: k=-
A - Aa s
where
A
= observed activity in Norit samples, cpm. S = computed activity in Norit samples, dpm. Ab = background activity, cpm. The two samples gave values for k of 0.146 cpd (counts per disintegration) and 0.157 cpd. The value used in this paper was taken as 0.15 cpd. Throughout the investigation the sample preparation, mounting, and counting techniques were identical to those used to determine the detection coefficient. No self-absorption correction was necessary since a linear relationship between the mass of the bacteria and their specific activity was obtained over the mass range (< 3 mg.) used in this study. RESULTS
AND
DISCUSSION
2-Amino-3-phenylbutanoic acid (APBA) was prepared by condensing (1 -bromoethyl)-
THIENYLALANINE
benzene with ethyl acetamidomalonate, followed by hydrolysis to the desired amino acid. The product was isolated as the hydrochloride salt, which had previously been prepared by another method (10). APBA inhibits the growth of L. dextranicum, and is competitively reversed by phenylalanine over a loo-fold range of concentrations, as indicated in Table I. The inhibition index (ratio of antagonist to metabolite) for half-maximal growth is about thirty. The presence of a methyl group on the P-carbon does not sterically prevent binding of the analog at the active site which complexes with the natural amino acid. Since it has been reported that a degree of planarity of the carbons attached to the p-carbon atom is essential for phenylalanine antagonism in L. dextralaicum (4), it may be assumed that the conformation of the inhibitory form of the analog is one in which the aromatic nucleus, the p-carbon and the carbon of the &methyl group are coplanar. Attempts to reverse the toxicity of APBA for L. dextranicum by tyrosine, shikimic acid or quinic acid, in a competitive manner, were all unsuccessful. These compounds do reverse the toxicity of low concentrations of APBA; however, higher concentrations of these natural products do not effect a reversal of higher concentrations of the analog. TABLE
I
REVERUL OF TOXICITY PHENYLBUTANOIC ACID 8086 BY DEXTRANICUM
OF Z-AMINO-% IN LEUCONOSTOC PHENYLALANINE
pg./??&
0
0.2
0.6
2
6
20
19
18
19
23 48 78
25 60
y0 Transmittance
m/ml.
0 0.06 0.2 0.6 2 6 20 60 100 600 1000
18 20 33 55
19
Incubated
19 hr. at 30°C.
20 34 67
19
21 46 73
TABLE
25 52 80
II
REVERSAL OF TOXICITY OF ~-AMINO-~ PHENYLBUTANOIC ACID IN LEUCONOSTOC DEXTRANICUM., 8086 BY TYROSINE Tyrosine Pg. /ml.
Z-Amino-Jpheny~~;danoic
0.2
0
K3./ml.
0 0.2 0.6 2 6 20 60 Incubated
.0.6
2
6
20
70 Transmittance
11 16 34 44
8
23 43
7
7
6
7
20 40 48
30 38 45
6 40 46
6 34 45
22 hr. at 30°C. TABLE
III
REVERSAL OF TOXICITY OF THIENYLALANINE ESCHERICHIA COLI W BY ~-AMINO-~PHENYLBUTANOIC ACID 2.Amino-3-phenylbutanoic !-%/ml.
@-2-
Thie$a$ine
0
6
20
IN
acid
60
200
32
34
‘% Transmittance
0 0.2 0.6 2 6 20 60 200 Incubated
Phenvlalanine 2.Amino-3%. pheny;k$anw
177
INCORPORATION
31 32 96 97
31
32
32 89
32 32 87
33 52 92
33 53 89
17 hr. at 37°C.
This effect is shown for tyrosine in Table II. The slight effect of tyrosine is probably the result of enhanced production of phenylalanine by the organism in which tyrosine diverts common precursors into the synthesis of phenylalanine. At high inhibitor concentrations, the organism may not be capable of synthesizing enough phenylalanine to reverse the toxicity. In contrast to the growth inhibitory properties of APBA for L. dextranicum, this analog is relatively nontoxic for E. c&i. However, it does reverse growth inhibition which is caused by the phenylalanine antagonist thienylalanine, as shown in Table III. The ratio of inhibitor (thienylalanine)
178
EDELSON
L FIG. 1. Effect
of washing
OF THIENYLALANINE-Cl* ESCRERICHIA COLI CELLS
Water Thienylalanine, 100 pg./ml. Phenylalanine, 100 pg./ml. 2-Amino-3-phenylbutanoic acid, 100 pg./ml.
4 OF
on cells
IV
DISPLACEMENT
Displacing agent
KEELEY
2 NUMBER
TABLE
AND
FROM
Molecules Loss, thienylalanine per cell x 10-s 7%
3.13 2.94 2.92 2.99
0 6.1 6.8 4.5
to reversing agent (APBA) is approximately unity over a 35-fold range of inhibitor concentrations, which suggests that the two synthetic amino acids are competing for a single enzymatic site. In an effort to elucidate this effect, the mode of action of APBA in preventing thienylalanine toxicity was investigated using radiochemical techniques. Cells of E. coli which were grown in a medium containing a subinhibitory level of /3-2-thienyl-nL-alanine-P-C14 were found to incorporate radioactivity, and repeated attempts to remove the labelled analog by washing with water were unsuccessful as shown in Fig. 1; the counting rate remains constant at about 1600 cpm. In addition, even when the bacteria which have been grown in thienylalanine-Cl4 are placed in phosphate buffer (pH 7.0) containing glucose and either unlabelled thienylalanine, APBA or phenylalanine, and incubated 20 hr. at
IO
ML.
grown
6
WASHES
in labelled
thienylalanine.
37”C., no more than about 7% of the radioactive analog may be displaced (Table IV). The concentration of the potential displacing agent in the latter system is 500-fold greater than the initial concentration of the labelled antagonist. The average number of molecules of inhibitor per bacteria was calculated by means of the formula: R = (NS/DkC), where R is the number of molecules per cell, N is Avogadro’s number, S is the specific activity of the bacteria in cpm per mg.; D is the specific activity of the inhibitor in dpm per GFW (6.7 X 1012dpm per GFW for this sample of thienylalanine-CY4), k is the detection coefficient (0.15 cpm per dpm under the experimental conditions), and C is the number of bacterial cells per milligram of dry weight (2.26 X log cells per mg. as determined by plate counts). Thus, most of the thienylalanine is incorporated in an essentially irreversible manner. These results are in accord with those of Rabinovitz et al. (II), who showed the incorporation of thienylalanine into prot,ein, using a different system. The incorporation of thienylalanine with time was studied to determine the rate at which equilibrium is established between the antagonist and the cells in a growing system. As shown in Fig. 2, the specific activity remains constant once the cells have left the logarithmic phase of growth. From the activity of the bacterial samples and the
THIENYLALANINE
179
INCORPORATION
initial .activity of the thienylalanine, the fraction of total activity incorporated into the bacteria can be determined. After 5 hr., when the specific activity is maximized, less than 2% of the activity is incorporated; whereas, when growth has stopped, after 15 hr., 16% of the total activity is in the bacterial cells. Therefore, in all radiochemical studies the cells were allowed to incubate a minimum of 18 hr. to assure maximum growth and incorporation. If E. coli cells are grown on a medium supplemented with labelled thienylalanine in the presence of either phenylalanine or APBA there is a decrease in incorporation of the labelled analog, as shown in Fig. 3.
TABLE THIENYLALANINE
V
INCORPORATION
ESCHERICHIA Thienylalanine r&/d.
Bacterial cell weight WT.
0
1.16 1.18 1.13 1.07 1.05 1.06 1.07 0.67 0.54 0.38 0.022
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 2.0 6.0
INTO
COLI
0 1147 3777 6431 8219 10,434 13,564 19,290 21,130 23,647 24,570
‘f--‘------%0.5 I// I:
TIME
FIG. 2. Growth
of cells
(HOURS)
on labelled
-4-
PHENYLALANINE
-
APBA
CONCENTRATION
FIG. 3. Repression presence
of a reversing
of incorporation agent.
of labelled
thienylalanine,
h(I/ML)
thienylalanine
when
E. coli cells are grown in the
180
EDELSON
AND
Therefore, in the reversal of thienylalanine toxicity by either of these two agents, their effect is to prevent, thienylalanine from combining with the cell in an irreversible manner. It is interesting to note that the synthetic reversing agent, APBA, is slightly more effective, at low concentrations, in preventing incorporation of the inhibitor than the natural reversing agent, phenylalanine. Since thienylalanine is incorporated into the bacterial cell, there must be a minimum concentration of thienylalanine within the cell which will be sufficient to inhibit completely the growth of this microorganism. The effect of increasing concentrations of this antagonist upon incorporation is tabulated in Table V. The size of the inoculum was increased 50-fold to give a measurable weight of bacterial cells. Under the conditions of this experiment 6 pg. per ml. was sufficient to inhibit the growth of E. coli. Thus, it follows that in order to induce virtually complete inhibition of growth, it requires approximately 6.7 X lo6 molecules of thienylalanine per E. coli cell. From these results, it is apparent that APBA does not inhibit the utilization of endogenously synthesized phenylalanine in E. coli, but would inhibit the utilization of exogenous phenylalanine. This is indicated by the ability of APBA to reverse the inhibitory effect, of thienylalanine, presumably by preventing an irreversible, essential step in the assimilation of thienylalanine into proteins. In contrast, for L. deztranicum,
KEELEY
APBA is a conventional antagonist of phenylalanine, blocking both biosynthetic processes and exogenous utilization. ACKNOWLEDGMENTS The authors are indebted to Mrs. A. S. Edelson, Miss J. M. Courmier, and Mr. L. A. Ortego for assistance with the microbial assays, and to Mr. C. Hedgcoth of the Clayton Foundation BioChemical Institute, University of Texas, Austin, for the elemental analyses. All melting points are uncorrected. REFERENCES 1. EDELSON, J., PAL, P. R., SKINNER, C. G., AND SHIVE, W., J. Am. Chem. Sot. ‘79, 5209 (1957) * 2. EDELSON, J., SKINNER, C. G., RAVEL, J. M., AND SHIVE, W., J. Am. Chem. Sot. 81, 5150 (1959). 3. PAL, P. R., SKINNER, C. G., DENNIS, R. L., AND SHIVE, W., J. Am. Chem. Sot. 78, 5116 (1956). 4. SMITH, L. C., SKINNER, C. G., AND SHIVE, W., Arch. Biochem. Biophys. 94, 443 (1961). 5. Fox, S. W., AND WARNER, C., J. Biol. Chem. 210, 119 (1954). 6. DUNN, F. W., Abstr. Am. Chem. Sot. 131st Meeting, 37C (Miami, Fla., .4pril 1957). 7. RAVEL, J. M., WOODS, L., FELSING, B. AND SHIVE, W., J. Biol. Chem. 206, 391 (1954). 8. DAVIS, B., AND MINGIOLI, E. S., J. Bacterial. 60, 17 (1950). 9. DUNN, F. W., RAVEL, J. M., AND SHIVE, W., J. Biol. Chem. 219, 810 (1956). 10. DARAPSKY, A., AND KOSTER, E., J. Prakl. Chem. 146, 287 (1936). 11. RABINOVITZ, M., OLSON, M. E., AND GREEKBERG, D. M., J. Riol. Chem. 210, 837 (1954).
OF
BIOCHEMISTRY
Inhibition
AND
103, 175-180 (1963)
BIOPHYSICS
of Thienylalanine
Incorporation
2-Amino-3-Phenylbutanoic JEROME From the Department
EDELSOIS’
of Chemistry,
Received
of Southwestern March
co/i by
Acid
AKD DEAN
University
in Escherichia
F. KEELEY Louisiana,
Lafayette,
Louisiana
28, 1963
2-Amino&phenylbutanoic acid was prepared via a typical malonic ester synthesis and shown to be an effective phenylalanine antagonist for Leuconostocdextranicum but not for Escherichia coli. However, it does competitively prevent the toxicity due to thienylalanine by inhibiting the incorporation of thienylalanine in E. coli. EXPERIMENTAL
INTRODUCTION
BIOLOGICAL ASSAYS
Studies of the biological effects of various phenylalanine analogs upon the growth of Leuconostoc dextranicum have shown that the steric configurations of the antagonists have a profound influence on their activities. The specificities of many nonaromatic analogs have been ascribed to the planarity and size of the group attached to the P-carbon of the alanine side chain (14). Substituent,s on the p-carbon of phenylalanine, e.g., fi-phenylserine (5), have also produced phenylalanine antagonists, and the present investigation was initiated in order to determine the effect upon biological activity of different substituents on the P-carbon
of the
natural
amino
For assays with L. dextranicum 8086, tyrosine and phenylalanine were omitted from a previously described amino acid medium (7), the salts A concentration was increased fourfold; the medium was further supplemented with 0.02 pg. per ml. pantethine and 0.2 pg. per ml. calcium pantothenate. The assays with E. coli W employed a salts-glucose medium (8). Mechanical details of this procedure have been reported in detail (9), except that, for cell counts, the medium was modified by the addition of agar to a final concentration of 2%. In all assays the amino acid analogs were dissolved in sterile water and added to sterile assay tubes without being heated. The amount of growth was determined turbidimetrically with a Bausch and Lomb Spectronic 20 calorimeter at 525 rnp, using distilled water to set 100% transmittance.
acid.
Accordingly, 2 - amino-3-phenylbutanoic acid (APBA) was prepared and its biological activity was investigated using L. dextranicum 8086 and Escherichia coli W as the assay organisms. Although not toxic for E’. co&i,APBA inhibits growth of L. dextranicum and its toxicity is competitively reversed by phenylalanine. In addition the toxicity of thienylalanine for E. coli, which may be reversed by analogs such or p-1-naphthylalanine
Wallace
Ethyl 2-Acetamido-d-CarbethoxyS-Phenylbutanoate
as p-tolylalanine
(6), may also be
relieved by APBA. 1 Present address: bury, New Jersey.
CHEMICAL SYNTHESIS
Laboratories,
Cran-
To a solution of 2.3 g. of sodium reacted with 200 ml. anhydrous ethanol, 20 g. of ethyl acetamidomalonate were added, followed by the addition of 18.5 g. of (I-bromoethyl)benzene. The reaction mixture was heated to reflux for 4 hr., and the inorganic salts which formed were removed by filtration. After pouring the alcoholic filtrate over ice, 13.5 g. of solids precipitated and were recrystallized from ethanol-water, m.p. 12P125”C.
175
176
EDELSON
Anal. Calcd. N, 4.50.
for CnH23NOb:
AND
N, 4.36. Found:
d-Amino-S-Phenylbutanoic
Acid
A mixture of 10 g. of ethyl 2-acetamido-2carbethoxy-3-phenylbutanoate and 100 ml. of 6 N hydrochloric acid was heated under reflux for 16 hr., and the resulting solution was evaporated to dryness in racuo. After two recrystalliaations from 6 N hydrochloric acid, 1.8 g. of sample was obtained, m.p. 200°C. dec.; reported m.p. 198”C., via a different method of preparation (10). Anal. Calcd. for CloH13N02.HCl: N, 6.50. Found: N, 6.31.
RADIOCHEMICAL
ASSAYS
Bacterial Studies Escherichia coli cells were grown in 10 ml. of salts-glucose medium (8) supplemented with 0.2 pg. per ml. of thienylalanine-j%Ci” added aseptically without heating, unless some other concentration is specified in the Tables or Figures. This concentration is not inhibitory and maximum growth is attained in about 15 hr. at 37°C. Incubation for a minimum of 18 hr. assures good growth. For washing effects and time studies, larger volumes were prepared and lo-ml. aliquots were taken after incubation. The cells were kept uniformly suspended by rapid stirring. The weight of cells was determined by turbidity measurements using a Beckman Model DU spectrophotometer. This was calibrated at 575 m by dilution of a culture of E. coli with medium, an aliquot of which was dried and weighed on a tared Millipore filter. Following separation of the suspended bacteria on a 47 mm. Millipore filter, type HA, the bacteria were subjected to three successive distilled water washes of 10, 10, and 80 ml. The samples were dried in an oven at 5960°C. and stored in a desiccator prior to counting. It was found that the filters could be conveniently held flat in a-inch stainless steel planchets during the drying and counting operations by means of g-inch segments of 1% inch iron pipe. The activity of the dried samples was measured by means of a Tracerlab Model FD-2 thin window (< 125 pg. per cm.2) gas flow counter employing methane-argon counting gas.
Determination
of the Detection
Coeficient
Ten PC. of a sample of P-2-thienyl-nn-alanine@-Cl4 (California Corporation for Biochemical Research) with a specific activity of 3.0 PC. per wmole was dissolved in 5.6 ml. of distilled water. The resulting solution, containing 100 pg. per ml. of the thienylalanine, was used as a stock solution for the entire study.
KEELEY
Twenty pl. of the thienylalanine stock solution was diluted to a volume of 50 ml. with distilled water. Four l.O-ml. aliquots of the diluted stock solution were evaporated to dryness in l-inch stainless steel planchets by means of a sample spinner-heat lamp assembly. The average activity, Ai, of the four samples was determined. Two loml. aliquots of the diluted stock solution were each treated with a sample of Norit weighing approximately 2 mg. The Norit was removed by filtration through a 47-mm., type HA Millipore filter, and dried without washing. The activity, A, of each sample was then determined. Four l.O-ml. aliquots of the filtrate from each of the Norit treated solutions were evaporated to dryness in 1 inch planchets and the average activity, A,, determined as before. The difference in average counting rates between the 1 ml. aliquot samples taken before and after Norit treatment was used to calculate the fraction, f, of the activity absorbed by the charcoal:
f=- Ai - A,
A< - Aa
where
Ai = initial activity of samples, cpm. Al = final activity of samples, cpm. & = From activity alanine sample k, was
background activity, cpm. the concentration of the solution, specific of thienylalanine, and fraction of thienylremoved, the activity, S, in each Norit was computed. The detection coefficient, then determined from the relationship: k=-
A - Aa s
where
A
= observed activity in Norit samples, cpm. S = computed activity in Norit samples, dpm. Ab = background activity, cpm. The two samples gave values for k of 0.146 cpd (counts per disintegration) and 0.157 cpd. The value used in this paper was taken as 0.15 cpd. Throughout the investigation the sample preparation, mounting, and counting techniques were identical to those used to determine the detection coefficient. No self-absorption correction was necessary since a linear relationship between the mass of the bacteria and their specific activity was obtained over the mass range (< 3 mg.) used in this study. RESULTS
AND
DISCUSSION
2-Amino-3-phenylbutanoic acid (APBA) was prepared by condensing (1 -bromoethyl)-
THIENYLALANINE
benzene with ethyl acetamidomalonate, followed by hydrolysis to the desired amino acid. The product was isolated as the hydrochloride salt, which had previously been prepared by another method (10). APBA inhibits the growth of L. dextranicum, and is competitively reversed by phenylalanine over a loo-fold range of concentrations, as indicated in Table I. The inhibition index (ratio of antagonist to metabolite) for half-maximal growth is about thirty. The presence of a methyl group on the P-carbon does not sterically prevent binding of the analog at the active site which complexes with the natural amino acid. Since it has been reported that a degree of planarity of the carbons attached to the p-carbon atom is essential for phenylalanine antagonism in L. dextralaicum (4), it may be assumed that the conformation of the inhibitory form of the analog is one in which the aromatic nucleus, the p-carbon and the carbon of the &methyl group are coplanar. Attempts to reverse the toxicity of APBA for L. dextranicum by tyrosine, shikimic acid or quinic acid, in a competitive manner, were all unsuccessful. These compounds do reverse the toxicity of low concentrations of APBA; however, higher concentrations of these natural products do not effect a reversal of higher concentrations of the analog. TABLE
I
REVERUL OF TOXICITY PHENYLBUTANOIC ACID 8086 BY DEXTRANICUM
OF Z-AMINO-% IN LEUCONOSTOC PHENYLALANINE
pg./??&
0
0.2
0.6
2
6
20
19
18
19
23 48 78
25 60
y0 Transmittance
m/ml.
0 0.06 0.2 0.6 2 6 20 60 100 600 1000
18 20 33 55
19
Incubated
19 hr. at 30°C.
20 34 67
19
21 46 73
TABLE
25 52 80
II
REVERSAL OF TOXICITY OF ~-AMINO-~ PHENYLBUTANOIC ACID IN LEUCONOSTOC DEXTRANICUM., 8086 BY TYROSINE Tyrosine Pg. /ml.
Z-Amino-Jpheny~~;danoic
0.2
0
K3./ml.
0 0.2 0.6 2 6 20 60 Incubated
.0.6
2
6
20
70 Transmittance
11 16 34 44
8
23 43
7
7
6
7
20 40 48
30 38 45
6 40 46
6 34 45
22 hr. at 30°C. TABLE
III
REVERSAL OF TOXICITY OF THIENYLALANINE ESCHERICHIA COLI W BY ~-AMINO-~PHENYLBUTANOIC ACID 2.Amino-3-phenylbutanoic !-%/ml.
@-2-
Thie$a$ine
0
6
20
IN
acid
60
200
32
34
‘% Transmittance
0 0.2 0.6 2 6 20 60 200 Incubated
Phenvlalanine 2.Amino-3%. pheny;k$anw
177
INCORPORATION
31 32 96 97
31
32
32 89
32 32 87
33 52 92
33 53 89
17 hr. at 37°C.
This effect is shown for tyrosine in Table II. The slight effect of tyrosine is probably the result of enhanced production of phenylalanine by the organism in which tyrosine diverts common precursors into the synthesis of phenylalanine. At high inhibitor concentrations, the organism may not be capable of synthesizing enough phenylalanine to reverse the toxicity. In contrast to the growth inhibitory properties of APBA for L. dextranicum, this analog is relatively nontoxic for E. c&i. However, it does reverse growth inhibition which is caused by the phenylalanine antagonist thienylalanine, as shown in Table III. The ratio of inhibitor (thienylalanine)
178
EDELSON
L FIG. 1. Effect
of washing
OF THIENYLALANINE-Cl* ESCRERICHIA COLI CELLS
Water Thienylalanine, 100 pg./ml. Phenylalanine, 100 pg./ml. 2-Amino-3-phenylbutanoic acid, 100 pg./ml.
4 OF
on cells
IV
DISPLACEMENT
Displacing agent
KEELEY
2 NUMBER
TABLE
AND
FROM
Molecules Loss, thienylalanine per cell x 10-s 7%
3.13 2.94 2.92 2.99
0 6.1 6.8 4.5
to reversing agent (APBA) is approximately unity over a 35-fold range of inhibitor concentrations, which suggests that the two synthetic amino acids are competing for a single enzymatic site. In an effort to elucidate this effect, the mode of action of APBA in preventing thienylalanine toxicity was investigated using radiochemical techniques. Cells of E. coli which were grown in a medium containing a subinhibitory level of /3-2-thienyl-nL-alanine-P-C14 were found to incorporate radioactivity, and repeated attempts to remove the labelled analog by washing with water were unsuccessful as shown in Fig. 1; the counting rate remains constant at about 1600 cpm. In addition, even when the bacteria which have been grown in thienylalanine-Cl4 are placed in phosphate buffer (pH 7.0) containing glucose and either unlabelled thienylalanine, APBA or phenylalanine, and incubated 20 hr. at
IO
ML.
grown
6
WASHES
in labelled
thienylalanine.
37”C., no more than about 7% of the radioactive analog may be displaced (Table IV). The concentration of the potential displacing agent in the latter system is 500-fold greater than the initial concentration of the labelled antagonist. The average number of molecules of inhibitor per bacteria was calculated by means of the formula: R = (NS/DkC), where R is the number of molecules per cell, N is Avogadro’s number, S is the specific activity of the bacteria in cpm per mg.; D is the specific activity of the inhibitor in dpm per GFW (6.7 X 1012dpm per GFW for this sample of thienylalanine-CY4), k is the detection coefficient (0.15 cpm per dpm under the experimental conditions), and C is the number of bacterial cells per milligram of dry weight (2.26 X log cells per mg. as determined by plate counts). Thus, most of the thienylalanine is incorporated in an essentially irreversible manner. These results are in accord with those of Rabinovitz et al. (II), who showed the incorporation of thienylalanine into prot,ein, using a different system. The incorporation of thienylalanine with time was studied to determine the rate at which equilibrium is established between the antagonist and the cells in a growing system. As shown in Fig. 2, the specific activity remains constant once the cells have left the logarithmic phase of growth. From the activity of the bacterial samples and the
THIENYLALANINE
179
INCORPORATION
initial .activity of the thienylalanine, the fraction of total activity incorporated into the bacteria can be determined. After 5 hr., when the specific activity is maximized, less than 2% of the activity is incorporated; whereas, when growth has stopped, after 15 hr., 16% of the total activity is in the bacterial cells. Therefore, in all radiochemical studies the cells were allowed to incubate a minimum of 18 hr. to assure maximum growth and incorporation. If E. coli cells are grown on a medium supplemented with labelled thienylalanine in the presence of either phenylalanine or APBA there is a decrease in incorporation of the labelled analog, as shown in Fig. 3.
TABLE THIENYLALANINE
V
INCORPORATION
ESCHERICHIA Thienylalanine r&/d.
Bacterial cell weight WT.
0
1.16 1.18 1.13 1.07 1.05 1.06 1.07 0.67 0.54 0.38 0.022
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 2.0 6.0
INTO
COLI
0 1147 3777 6431 8219 10,434 13,564 19,290 21,130 23,647 24,570
‘f--‘------%0.5 I// I:
TIME
FIG. 2. Growth
of cells
(HOURS)
on labelled
-4-
PHENYLALANINE
-
APBA
CONCENTRATION
FIG. 3. Repression presence
of a reversing
of incorporation agent.
of labelled
thienylalanine,
h(I/ML)
thienylalanine
when
E. coli cells are grown in the
180
EDELSON
AND
Therefore, in the reversal of thienylalanine toxicity by either of these two agents, their effect is to prevent, thienylalanine from combining with the cell in an irreversible manner. It is interesting to note that the synthetic reversing agent, APBA, is slightly more effective, at low concentrations, in preventing incorporation of the inhibitor than the natural reversing agent, phenylalanine. Since thienylalanine is incorporated into the bacterial cell, there must be a minimum concentration of thienylalanine within the cell which will be sufficient to inhibit completely the growth of this microorganism. The effect of increasing concentrations of this antagonist upon incorporation is tabulated in Table V. The size of the inoculum was increased 50-fold to give a measurable weight of bacterial cells. Under the conditions of this experiment 6 pg. per ml. was sufficient to inhibit the growth of E. coli. Thus, it follows that in order to induce virtually complete inhibition of growth, it requires approximately 6.7 X lo6 molecules of thienylalanine per E. coli cell. From these results, it is apparent that APBA does not inhibit the utilization of endogenously synthesized phenylalanine in E. coli, but would inhibit the utilization of exogenous phenylalanine. This is indicated by the ability of APBA to reverse the inhibitory effect, of thienylalanine, presumably by preventing an irreversible, essential step in the assimilation of thienylalanine into proteins. In contrast, for L. deztranicum,
KEELEY
APBA is a conventional antagonist of phenylalanine, blocking both biosynthetic processes and exogenous utilization. ACKNOWLEDGMENTS The authors are indebted to Mrs. A. S. Edelson, Miss J. M. Courmier, and Mr. L. A. Ortego for assistance with the microbial assays, and to Mr. C. Hedgcoth of the Clayton Foundation BioChemical Institute, University of Texas, Austin, for the elemental analyses. All melting points are uncorrected. REFERENCES 1. EDELSON, J., PAL, P. R., SKINNER, C. G., AND SHIVE, W., J. Am. Chem. Sot. ‘79, 5209 (1957) * 2. EDELSON, J., SKINNER, C. G., RAVEL, J. M., AND SHIVE, W., J. Am. Chem. Sot. 81, 5150 (1959). 3. PAL, P. R., SKINNER, C. G., DENNIS, R. L., AND SHIVE, W., J. Am. Chem. Sot. 78, 5116 (1956). 4. SMITH, L. C., SKINNER, C. G., AND SHIVE, W., Arch. Biochem. Biophys. 94, 443 (1961). 5. Fox, S. W., AND WARNER, C., J. Biol. Chem. 210, 119 (1954). 6. DUNN, F. W., Abstr. Am. Chem. Sot. 131st Meeting, 37C (Miami, Fla., .4pril 1957). 7. RAVEL, J. M., WOODS, L., FELSING, B. AND SHIVE, W., J. Biol. Chem. 206, 391 (1954). 8. DAVIS, B., AND MINGIOLI, E. S., J. Bacterial. 60, 17 (1950). 9. DUNN, F. W., RAVEL, J. M., AND SHIVE, W., J. Biol. Chem. 219, 810 (1956). 10. DARAPSKY, A., AND KOSTER, E., J. Prakl. Chem. 146, 287 (1936). 11. RABINOVITZ, M., OLSON, M. E., AND GREEKBERG, D. M., J. Riol. Chem. 210, 837 (1954).