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Comparative inhibitory effects of glutamic and cysteic acids on aspartic acid utilization
Comparative Inhibitory Effects of Glutamic and Cysteic Acids on Aspartic Acid Utilization’ Joanne M. Ravel,2 Barbara Felsing and William From
the Biochenlical Institute and the Department Texas and the Clayton Foundation for Received
June
of Chemistry, Research, Austin,
Shive The University Texas
of
21, 1954
In Leuconostoc dextranicum 8086, glutamic acid inhibits the utilization of aspartic acid in the biosynthesis of threonine (l), and in Lactobacillus arabinosus 17-5, cysteic acid prevents the conversion of aspartic acid to pyrimidines (2), threonine, and lysine (3). Further studies indicate that these aspartic acid analogs prevent the utilization of aspartic acid in both organisms but exert their effects in different manners such as to indicate a difference in the site of the inhibitory action of glutamic acid and cysteic acid. EXPERIMENTAL
Materials The amino acids and other compounds were obtained from sources unless otherwise indicated. The authors are indebted for preparations of L-cysteic acid and to Dr. E. E. Snell osalacetic acid.
standard commercial to Dr. C. G. Skinner for a preparation of
Methods Cultures of Leuc. dextranicum 8086 and L. arabinosus 17-5 were maintained by conventional bacteriological practices. The assay procedure and medium were the same as previously described (3) with the following exceptions: glutamic acid, threonine, lysine, and uracil were omitted from the basal medium; the biotin concentration was lowered to 5 mpg./5 ml. assay tube; the purine supplement was I From a thesis submitted by Joanne University of Texas, in partial fulfillment Doctor of Philosophy, May, 1954. * Eli Lilly and Co. Predoctoral Fellow.
M.
541
Ravel to the Graduate of the requirements for
School, The the degree of
542
RAVEL,
FELSING
AND
SHIVE
decreased to 0.8 ml./100 ml. of basal medium; 200 pg. of L-glutamine was added aseptically to each assay tube after autoclaving; and for assays with Leuc. deztranicum 8086, aspartic acid was omitted from the basal medium, and the concentration of Salts A was increased fourfold.
Results In Leuc. dextranicum 8086, cysteic acid competitively inhibits the utilization of aspartic acid, the ratio of cysteic acid to aspartic acid necessary for maximal inhibition of growth being slightly greater than 2000 as indicated in Table I. Similar to previously reported data (l), separate experiments with L. arabinosus 17-5 gave ratios of cysteic acid to aspartic TABLE Competitive
Inhibition Leuc.
I-Cysteic
acid
mgJ5 ml.
d&&cum
of Aspartic
DL-Asyrtlc
Acid
8086a
GalvanpmeJer readmgs
pp.15 ml.
0
0
1 1 1 1 1
0 0.5 1 2 5
I
Utilization
by
nL-Glutamic acid mg./5 ml.
53 17 28 30 41 47
2 2 2 2 2
0 1 2 5 10
10 15 27 43 48
5 5 5 5 5
0 2 5 10 20
6 15 23 49 50
10 0 9 10 5 17 10 36 10 41 10 20 52 10 50 0 Incubated 26 hr. at 30”. b Incubated 22 hr. at 30”. c A measure of culture turbidity;
and. Cystic
L. orabinosus m-f&artx
,mb
Acids
Galvanfxneter readmgsC
pg.15 ml.
0
0
5 5 5 5 5
0 20 50 100 200
6 7 18 35 75
10 10 10 10
0 50 100 200
5 7 30 74
20
0
20 20 20
100 200 500
3 11 53 77
50
50 50 50
distilled
Glutamic
0
100 200 500
64
3 2 5 58
water reads 0, an opaque object 100.
INHIBITION
OF
ASPARTIC
ACID
TJTILIZATION
543
acid of 20-40 for similar degrees of inhibition over the same concentration range. On the other hand, glutamic acid prevents competitively the utilization of aspartic acid in Leuc. dextranicum 8086 more effectively than in L. arabinosus 17-5. The ratio of glutamic acid to aspartic acid necessary for maximal growth inhibition is 50 for Leuc. dextranicum 8086 in this medium and is of the same magnitude as the ratio previously reported for a slightly different medium (1). As indicated in Table I, the ratio is approximately 200 for L. arabinosus 17-5 under similar conditions. Previous reports (2, 3) have indicated that, for L. arabinosus 17-5, supplements of pyrimidines, threonine, and lysine each increase the amount of cysteic acid necessary for a defined inhibition and that combinations of two of the metabolites are more effective than any one, and the combination of all three exerts a greater effect than any combination of two. Similar results are obtained with glutamic acid as the inhibitor as indicated in Table II, but a combination of lysine and threonine is relatively more effective in reversing glutamic acid toxicity than cysteic acid toxicity, and the effects of uracil alone or in combinations is very slight. By contrast, results obtained with Leuc. dextranicum 8086 indicate that lysine has only slight effects on cysteic acid toxicity and no appreciable effect on glutamic acid inhibition. A combination of uracil and threonine completely prevents the toxicity of glutamic acid in the absence of supplements of lysine. Cysteic acid prevents maximal but not suboptimal growth in the absence of lysine, but bicarbonate contamination of neutralized inhibitor solutions may account for the appearance of suboptimal growth since bicarbonate reverses the toxicity of cysteic acid in a competitive manner (Table III). It. is interesting that in the medium containing 200 pg. of glutamic acid/5 ml., Leuc. dextranicum 8086 requires either aspartic acid or both a pyrimidine and threonine for growth, but in the absence of glutamic acid the organism does not require supplements of either aspartic acid or pyrimidines and threonine for growth. The variations in the effects of these reversing agents suggested the possibility that cysteic acid and glutamic acid may act at different sites, and this possibility was further tested by the relative effects of bicarbonate, pyruvate, and oxalacetate on the two inhibitors. For Leuc. dextranicum 8086, all three of these compounds prevent the toxicity of cysteic acid in a competitive manner as indicated in Table III; however, glutamic acid even at high concentrations does not inhibit growth stimu-
544
RAVEL,
FELSING
AND
TABLE Effects
of Threonine,
Lysine
Inhibitor m,-Glutamic acid mg./5 ml.
and
L
Uracil
II
on the Toxicity Acids
T
su plement~ Ga Pvanometer readings u L, u
L. arabinosus 0 0.2 0.5 2 2 5 10 20
55 41 18 18 12 10
61
55
55 38 27 11
52 37 12
Leuc. 0 0.01 0.02 0.05 0.1 0.2 1.0 5.0
56 35 23 15 9
52 26 13 10 13
of
Glutamic
and
Cysteic
L T
T, U
L T, U
68
71
72
75
38 20 10
69 68 66 70 58
65 47 35 24 23
80 80 79 79 77
59
69
65
41 25 20
56 57 58 59
17-56 59 57 26 28 12 11
dextranicum 50 44 33 19 10 8
SHIVE
8086” 59 60 51 16 8 1
64
34 22 13
65 66
L-Cyst&c acid mg./5 ml.
0 0.2 0.5 1.0 2 5 20
53 33 15 4 1
51 39 20 3 1
53 49 30 13 18 12
59 53 55 34 6
65
57
60
62
55 23 1 1
49 47 31 9
56 54 54 33 42
62 59 61 58 55
0 Supplements of nn-lysine (L), nn-threonine (T), and uracil (U) (50 pg. of each amino acid and 10 #g. uracil/5 ml. for Leuc. dextranicum 8086 and 200 pg. each amino acid and 20 pg. uracil for L. arabinosus 17-5) added as indicated. b Incubated 21 hr. at 30”. c Incubated 25 hr. at 30”.
lation by bicarbonate, pyruvate, and oxalacetate. Pyruvate does not affect the toxicity of either glutamic acid or cysteic acid for L. arabinosus, but both bicarbonate and oxalacetate exert effects analogous to those observed with Leuc. dextranicum 8086. Oxalacetate is approximately one-tenth as effective as aspartic acid in preventing cysteic acid toxicity for both organisms.
INHIBITION
OF
ASPARTIC
SCID
TABLE Comparative
Effects
Leuc.
Inhibit .or L-Cyst eic acid
I nl.
0 2
2 2 2
10 10 10 10 10
Oxalacetatec~d
0 0 50 100 206
55 11 13 25 53
55 15 42 57
55 15 45 57
0 50 100 200 500
4
2 18 42 49 53 7 30 38 49 53
3 11 50 55
of
17-F Bicarbon&e
Supplements
OXC31acetateC
G &ammeterreadings mg./5 ml.
!-tg./j
ml.
0 0 500 1000 2000
64 22 31 50 69
64 22 20 38 50
2 32 47 51 56
0 500 1000 2000 5000
14 20 40 56
l-4 16 23 39 60
7 31 45 59 58
0 500 1000
6 7 21 45
6 8 8 12 40
500 1000 2000
5 40 53
5 30 53
500 1000 2000
8 32 58
8 33 58
DL-GIU mic ac mg.15
Inhibitor L-Cysteic acid
readings
~g./jml.
13 28 51
on the Toxicities
L. arabinosus
Supplements
0 100 200 500 1000
and Bicarbonate
Glutamic Acid
8086”
Galvanometer %?.I5
III
of Oralacetate, Pyruvate Custeic Acid and dextranicurn
545
UTILIZrlTION
0 1 1 1 1
5000 oL-Glutamic acid6
1
mg.15 ml.
0.: 0.: 0.: 0.:
0 50 100 200
8 19 30 53
8 33 49 59
8 39 54 62
1.C 1.C 1.C 1.C
0 50 100 200
12 18 25 51
12 36 56 61
12 29 50 67
0 0 0
50
50 50
0 Incubated 21 hr. at 30”. b Added aseptically to auto&wed medium. c Added aseptically to two portions, one at outset hr. of incubation. d Separate experiment with pyruvate as control. e Separate experiment, aspartic acid omitted from
of experiment,
basal
medium.
one
after
16
546
RAVEL,
FELSING
AND
TABLE
Synergistic
IV
Action of Cysteic Acid and Glutamic Acid
organism,L.
Test
arabinosus
17-5,
incubated
L-Cysteic Dr.-Glutamic
acid
SHIVE
None
1
0.05
/
0.1
acid, 1
0.2
Galvanometer
-___
-
0 0.05 0.1 0.2 0.5 1 2 5
55
49 49 49 47 47 35 12
55 52 44 37 7 -
-
47 45 45 45 30 13 7
-
at 30”.
mg./5 ml. /
I
0.5
1
1
)
2
readings
-
mg.f5 ml.
20 hr.
45 45 43 41 27 10
-
35 29 27 19 9
23
13
-
If two steps in sequence are inhibited, there is a possibility for synergistic effects of combinations of the two inhibitors. Such effects were indeed obtained with combinations of cysteic acid and glutamic acid in L. arabinosus 17-5 as indicated in Table IV. As little as one-tenth the amount of cysteic acid and one-fifth the amount of glutamic acid necessary for inhibition of growth gave a similar degree of inhibition in combinations. DISCUSSION
Glutamic acid inhibits the utilization of aspartic acid in reactions concerned with the biosynthesis of pyrimidines as well as threonine in Leuc. dextranicum 8086 and with the biosynthesis of lysine, threonine and to some extent pyrimidines in L. arabinosus 17-5. Cysteic acid also prevents the utilization of aspartic acid in reactions concerning the biosynthesis of all three products of aspartic acid. The synergistic action of glutamic acid and cysteic acid indicates that different modes of action of the two inhibitors are involved, presumably at two different steps in the utilization of aspartic acid. The possibility that one inhibitor prevents the transfer of aspartic acid into the cell and the other inhibits the utilization of aspartic acid within the cell can be excluded by the fact that both cysteic acid and glutamic acid are inhibitory under conditions such that Leuc. dextranicum 8086 is
INHIRITION
OF
.4SI’.4RTIC
ACID
UTILIZATIOK
547
synthesizing its aspartic acid. Since glutamic acid and cysteic acid apparently inhibit at different sites in utilization of aspartic acid, certain common intermediates appear to be involved in the biosynthesis of threonine, lysine, and pyrimidines. After a role of aspartic acid in the biosynthesis of threonine which was replaced by homoserine or its keto analog was reported (3), p-asparty1 phosphate was implicated in the conversion of aspartic acid to homoserine (4). The possibility that P-aspartyl phosphate is involved in the above biosyntheses appears likely, but other conjugates such as @-asparty coenzyme A may also have roles in aspartic acid utilization.3 Since growth stimulated by oxalacetate and its precursors is not inhibited by glutamic acid but is inhibited competitively by cysteic acid, these substances appear to be utilized by a route not involving aspartic acid per se. However, oxalacetate and its precursors are required in relatively high concentrations to exert such effects. The concurrent formation of adenosine triphosphate from adenosine diphosphate with the carboxylation of phosphoryl erfolpyruvate (5) suggests the possibility of a phosphorylated oxalacetate as an intermediate which on the basis of recent experiments could not be the phosphorylated enol but could be an acyl phosphate (6). Such a mechanism could allow the formation of conjugated forms of aspartic acid from precursors of oxalacetate without involving aspartic acid per se. If the transfer of phosphate from the intermediate to adenosine diphosphate is reversible, oxalacetate could be phosphorylated before transamination or other reactions forming aspartic acid derivatives, without involvement of aspartic acid. On the other hand, the amount of oxalacetate and its precursors necessary for growth stimulation may exceed the amount necessary for reversal of the inhibition by virtue of strong sparing effects of these substances on biosynthetic aspartic acid. In regard to the nutrition of Leuc. dextranicum 8086, either aspartic acid or uracil is required for growth in a medium containing all of the amino acids with exception of aspartic acid; however, substitution of glutamine for glutamic acid in the medium allows growth of the organism in the absence of aspartic acid and uracil. Thus, as with the threonine or aspartic acid requirement (l), the requirement for either aspartic arid or uracil results from an inhibition rather than the lack of an enzymatic process. 3 Since this manuscript coenzyme A in @-asparty Hirsch, M-L., Wiesendanger,
was submitted for publication, evidence for a role of phosphate synthesis has been presented (Cohen, G. N., S. B., and Nisman, B., Contpt. rend. 238,1746 (1954)).
548
RAVEL,
FELSING
AND
SHIVE
SUMMARY
Glutamic acid inhibits the utilization of aspartic acid in reactions concerned with the biosynthesis of pyrimidines as well as threonine in Leuconostoc dextranicum 8086 and with the biosynthesis of lysine, threonine, and to some extent pyrimidines in Lactobacillus arabinosus 17-5. Cysteic acid prevents the utilization of aspartic acid in reactions involved in the biosynthesis of these end products in both organisms. Since glutamic acid does not prevent the utilization of precursors of aspartic acid such as bicarbonate and oxalacetate which reverse cysteic acid inhibition competitively, different points of action with cysteic acid probably acting subsequent to glutamic acid are involved. The synergistic effects of combinations of the two inhibitors on L. arabinosus 17-5 offers further evidence for the. involvement of a sequence of inhibitions. REFERENCES 1. RAVEL, J. M., FELSING, B., AND SHIVE, W., J. Biol. Chem. 206, 791 (1954). 2. WOODS, L., RAVEL, J. M., AND SHIVE, W., J. Biol. Chem. 209,559 (1954). 3. RAVEL, J. M., WOODS, L., FELSING, B., AND SHIVE, W., J. Biol. Chem. 206, 391 (1954). 4. BLACK, S., AND GRAY, N. M., J. Am,. Chem. 5. BANDURSKI, R. S., AND GREINER, C. M., J. 6. VENNESLAND, B., TCHEN, T. T., AND LOEWUS, (1954).
Sot. 76, 5766 (1953). Biol. Chem. 204, 781 (1953). F. A., J. Am. Chem. Sot. 76,3358
the Biochenlical Institute and the Department Texas and the Clayton Foundation for Received
June
of Chemistry, Research, Austin,
Shive The University Texas
of
21, 1954
In Leuconostoc dextranicum 8086, glutamic acid inhibits the utilization of aspartic acid in the biosynthesis of threonine (l), and in Lactobacillus arabinosus 17-5, cysteic acid prevents the conversion of aspartic acid to pyrimidines (2), threonine, and lysine (3). Further studies indicate that these aspartic acid analogs prevent the utilization of aspartic acid in both organisms but exert their effects in different manners such as to indicate a difference in the site of the inhibitory action of glutamic acid and cysteic acid. EXPERIMENTAL
Materials The amino acids and other compounds were obtained from sources unless otherwise indicated. The authors are indebted for preparations of L-cysteic acid and to Dr. E. E. Snell osalacetic acid.
standard commercial to Dr. C. G. Skinner for a preparation of
Methods Cultures of Leuc. dextranicum 8086 and L. arabinosus 17-5 were maintained by conventional bacteriological practices. The assay procedure and medium were the same as previously described (3) with the following exceptions: glutamic acid, threonine, lysine, and uracil were omitted from the basal medium; the biotin concentration was lowered to 5 mpg./5 ml. assay tube; the purine supplement was I From a thesis submitted by Joanne University of Texas, in partial fulfillment Doctor of Philosophy, May, 1954. * Eli Lilly and Co. Predoctoral Fellow.
M.
541
Ravel to the Graduate of the requirements for
School, The the degree of
542
RAVEL,
FELSING
AND
SHIVE
decreased to 0.8 ml./100 ml. of basal medium; 200 pg. of L-glutamine was added aseptically to each assay tube after autoclaving; and for assays with Leuc. deztranicum 8086, aspartic acid was omitted from the basal medium, and the concentration of Salts A was increased fourfold.
Results In Leuc. dextranicum 8086, cysteic acid competitively inhibits the utilization of aspartic acid, the ratio of cysteic acid to aspartic acid necessary for maximal inhibition of growth being slightly greater than 2000 as indicated in Table I. Similar to previously reported data (l), separate experiments with L. arabinosus 17-5 gave ratios of cysteic acid to aspartic TABLE Competitive
Inhibition Leuc.
I-Cysteic
acid
mgJ5 ml.
d&&cum
of Aspartic
DL-Asyrtlc
Acid
8086a
GalvanpmeJer readmgs
pp.15 ml.
0
0
1 1 1 1 1
0 0.5 1 2 5
I
Utilization
by
nL-Glutamic acid mg./5 ml.
53 17 28 30 41 47
2 2 2 2 2
0 1 2 5 10
10 15 27 43 48
5 5 5 5 5
0 2 5 10 20
6 15 23 49 50
10 0 9 10 5 17 10 36 10 41 10 20 52 10 50 0 Incubated 26 hr. at 30”. b Incubated 22 hr. at 30”. c A measure of culture turbidity;
and. Cystic
L. orabinosus m-f&artx
,mb
Acids
Galvanfxneter readmgsC
pg.15 ml.
0
0
5 5 5 5 5
0 20 50 100 200
6 7 18 35 75
10 10 10 10
0 50 100 200
5 7 30 74
20
0
20 20 20
100 200 500
3 11 53 77
50
50 50 50
distilled
Glutamic
0
100 200 500
64
3 2 5 58
water reads 0, an opaque object 100.
INHIBITION
OF
ASPARTIC
ACID
TJTILIZATION
543
acid of 20-40 for similar degrees of inhibition over the same concentration range. On the other hand, glutamic acid prevents competitively the utilization of aspartic acid in Leuc. dextranicum 8086 more effectively than in L. arabinosus 17-5. The ratio of glutamic acid to aspartic acid necessary for maximal growth inhibition is 50 for Leuc. dextranicum 8086 in this medium and is of the same magnitude as the ratio previously reported for a slightly different medium (1). As indicated in Table I, the ratio is approximately 200 for L. arabinosus 17-5 under similar conditions. Previous reports (2, 3) have indicated that, for L. arabinosus 17-5, supplements of pyrimidines, threonine, and lysine each increase the amount of cysteic acid necessary for a defined inhibition and that combinations of two of the metabolites are more effective than any one, and the combination of all three exerts a greater effect than any combination of two. Similar results are obtained with glutamic acid as the inhibitor as indicated in Table II, but a combination of lysine and threonine is relatively more effective in reversing glutamic acid toxicity than cysteic acid toxicity, and the effects of uracil alone or in combinations is very slight. By contrast, results obtained with Leuc. dextranicum 8086 indicate that lysine has only slight effects on cysteic acid toxicity and no appreciable effect on glutamic acid inhibition. A combination of uracil and threonine completely prevents the toxicity of glutamic acid in the absence of supplements of lysine. Cysteic acid prevents maximal but not suboptimal growth in the absence of lysine, but bicarbonate contamination of neutralized inhibitor solutions may account for the appearance of suboptimal growth since bicarbonate reverses the toxicity of cysteic acid in a competitive manner (Table III). It. is interesting that in the medium containing 200 pg. of glutamic acid/5 ml., Leuc. dextranicum 8086 requires either aspartic acid or both a pyrimidine and threonine for growth, but in the absence of glutamic acid the organism does not require supplements of either aspartic acid or pyrimidines and threonine for growth. The variations in the effects of these reversing agents suggested the possibility that cysteic acid and glutamic acid may act at different sites, and this possibility was further tested by the relative effects of bicarbonate, pyruvate, and oxalacetate on the two inhibitors. For Leuc. dextranicum 8086, all three of these compounds prevent the toxicity of cysteic acid in a competitive manner as indicated in Table III; however, glutamic acid even at high concentrations does not inhibit growth stimu-
544
RAVEL,
FELSING
AND
TABLE Effects
of Threonine,
Lysine
Inhibitor m,-Glutamic acid mg./5 ml.
and
L
Uracil
II
on the Toxicity Acids
T
su plement~ Ga Pvanometer readings u L, u
L. arabinosus 0 0.2 0.5 2 2 5 10 20
55 41 18 18 12 10
61
55
55 38 27 11
52 37 12
Leuc. 0 0.01 0.02 0.05 0.1 0.2 1.0 5.0
56 35 23 15 9
52 26 13 10 13
of
Glutamic
and
Cysteic
L T
T, U
L T, U
68
71
72
75
38 20 10
69 68 66 70 58
65 47 35 24 23
80 80 79 79 77
59
69
65
41 25 20
56 57 58 59
17-56 59 57 26 28 12 11
dextranicum 50 44 33 19 10 8
SHIVE
8086” 59 60 51 16 8 1
64
34 22 13
65 66
L-Cyst&c acid mg./5 ml.
0 0.2 0.5 1.0 2 5 20
53 33 15 4 1
51 39 20 3 1
53 49 30 13 18 12
59 53 55 34 6
65
57
60
62
55 23 1 1
49 47 31 9
56 54 54 33 42
62 59 61 58 55
0 Supplements of nn-lysine (L), nn-threonine (T), and uracil (U) (50 pg. of each amino acid and 10 #g. uracil/5 ml. for Leuc. dextranicum 8086 and 200 pg. each amino acid and 20 pg. uracil for L. arabinosus 17-5) added as indicated. b Incubated 21 hr. at 30”. c Incubated 25 hr. at 30”.
lation by bicarbonate, pyruvate, and oxalacetate. Pyruvate does not affect the toxicity of either glutamic acid or cysteic acid for L. arabinosus, but both bicarbonate and oxalacetate exert effects analogous to those observed with Leuc. dextranicum 8086. Oxalacetate is approximately one-tenth as effective as aspartic acid in preventing cysteic acid toxicity for both organisms.
INHIBITION
OF
ASPARTIC
SCID
TABLE Comparative
Effects
Leuc.
Inhibit .or L-Cyst eic acid
I nl.
0 2
2 2 2
10 10 10 10 10
Oxalacetatec~d
0 0 50 100 206
55 11 13 25 53
55 15 42 57
55 15 45 57
0 50 100 200 500
4
2 18 42 49 53 7 30 38 49 53
3 11 50 55
of
17-F Bicarbon&e
Supplements
OXC31acetateC
G &ammeterreadings mg./5 ml.
!-tg./j
ml.
0 0 500 1000 2000
64 22 31 50 69
64 22 20 38 50
2 32 47 51 56
0 500 1000 2000 5000
14 20 40 56
l-4 16 23 39 60
7 31 45 59 58
0 500 1000
6 7 21 45
6 8 8 12 40
500 1000 2000
5 40 53
5 30 53
500 1000 2000
8 32 58
8 33 58
DL-GIU mic ac mg.15
Inhibitor L-Cysteic acid
readings
~g./jml.
13 28 51
on the Toxicities
L. arabinosus
Supplements
0 100 200 500 1000
and Bicarbonate
Glutamic Acid
8086”
Galvanometer %?.I5
III
of Oralacetate, Pyruvate Custeic Acid and dextranicurn
545
UTILIZrlTION
0 1 1 1 1
5000 oL-Glutamic acid6
1
mg.15 ml.
0.: 0.: 0.: 0.:
0 50 100 200
8 19 30 53
8 33 49 59
8 39 54 62
1.C 1.C 1.C 1.C
0 50 100 200
12 18 25 51
12 36 56 61
12 29 50 67
0 0 0
50
50 50
0 Incubated 21 hr. at 30”. b Added aseptically to auto&wed medium. c Added aseptically to two portions, one at outset hr. of incubation. d Separate experiment with pyruvate as control. e Separate experiment, aspartic acid omitted from
of experiment,
basal
medium.
one
after
16
546
RAVEL,
FELSING
AND
TABLE
Synergistic
IV
Action of Cysteic Acid and Glutamic Acid
organism,L.
Test
arabinosus
17-5,
incubated
L-Cysteic Dr.-Glutamic
acid
SHIVE
None
1
0.05
/
0.1
acid, 1
0.2
Galvanometer
-___
-
0 0.05 0.1 0.2 0.5 1 2 5
55
49 49 49 47 47 35 12
55 52 44 37 7 -
-
47 45 45 45 30 13 7
-
at 30”.
mg./5 ml. /
I
0.5
1
1
)
2
readings
-
mg.f5 ml.
20 hr.
45 45 43 41 27 10
-
35 29 27 19 9
23
13
-
If two steps in sequence are inhibited, there is a possibility for synergistic effects of combinations of the two inhibitors. Such effects were indeed obtained with combinations of cysteic acid and glutamic acid in L. arabinosus 17-5 as indicated in Table IV. As little as one-tenth the amount of cysteic acid and one-fifth the amount of glutamic acid necessary for inhibition of growth gave a similar degree of inhibition in combinations. DISCUSSION
Glutamic acid inhibits the utilization of aspartic acid in reactions concerned with the biosynthesis of pyrimidines as well as threonine in Leuc. dextranicum 8086 and with the biosynthesis of lysine, threonine and to some extent pyrimidines in L. arabinosus 17-5. Cysteic acid also prevents the utilization of aspartic acid in reactions concerning the biosynthesis of all three products of aspartic acid. The synergistic action of glutamic acid and cysteic acid indicates that different modes of action of the two inhibitors are involved, presumably at two different steps in the utilization of aspartic acid. The possibility that one inhibitor prevents the transfer of aspartic acid into the cell and the other inhibits the utilization of aspartic acid within the cell can be excluded by the fact that both cysteic acid and glutamic acid are inhibitory under conditions such that Leuc. dextranicum 8086 is
INHIRITION
OF
.4SI’.4RTIC
ACID
UTILIZATIOK
547
synthesizing its aspartic acid. Since glutamic acid and cysteic acid apparently inhibit at different sites in utilization of aspartic acid, certain common intermediates appear to be involved in the biosynthesis of threonine, lysine, and pyrimidines. After a role of aspartic acid in the biosynthesis of threonine which was replaced by homoserine or its keto analog was reported (3), p-asparty1 phosphate was implicated in the conversion of aspartic acid to homoserine (4). The possibility that P-aspartyl phosphate is involved in the above biosyntheses appears likely, but other conjugates such as @-asparty coenzyme A may also have roles in aspartic acid utilization.3 Since growth stimulated by oxalacetate and its precursors is not inhibited by glutamic acid but is inhibited competitively by cysteic acid, these substances appear to be utilized by a route not involving aspartic acid per se. However, oxalacetate and its precursors are required in relatively high concentrations to exert such effects. The concurrent formation of adenosine triphosphate from adenosine diphosphate with the carboxylation of phosphoryl erfolpyruvate (5) suggests the possibility of a phosphorylated oxalacetate as an intermediate which on the basis of recent experiments could not be the phosphorylated enol but could be an acyl phosphate (6). Such a mechanism could allow the formation of conjugated forms of aspartic acid from precursors of oxalacetate without involving aspartic acid per se. If the transfer of phosphate from the intermediate to adenosine diphosphate is reversible, oxalacetate could be phosphorylated before transamination or other reactions forming aspartic acid derivatives, without involvement of aspartic acid. On the other hand, the amount of oxalacetate and its precursors necessary for growth stimulation may exceed the amount necessary for reversal of the inhibition by virtue of strong sparing effects of these substances on biosynthetic aspartic acid. In regard to the nutrition of Leuc. dextranicum 8086, either aspartic acid or uracil is required for growth in a medium containing all of the amino acids with exception of aspartic acid; however, substitution of glutamine for glutamic acid in the medium allows growth of the organism in the absence of aspartic acid and uracil. Thus, as with the threonine or aspartic acid requirement (l), the requirement for either aspartic arid or uracil results from an inhibition rather than the lack of an enzymatic process. 3 Since this manuscript coenzyme A in @-asparty Hirsch, M-L., Wiesendanger,
was submitted for publication, evidence for a role of phosphate synthesis has been presented (Cohen, G. N., S. B., and Nisman, B., Contpt. rend. 238,1746 (1954)).
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RAVEL,
FELSING
AND
SHIVE
SUMMARY
Glutamic acid inhibits the utilization of aspartic acid in reactions concerned with the biosynthesis of pyrimidines as well as threonine in Leuconostoc dextranicum 8086 and with the biosynthesis of lysine, threonine, and to some extent pyrimidines in Lactobacillus arabinosus 17-5. Cysteic acid prevents the utilization of aspartic acid in reactions involved in the biosynthesis of these end products in both organisms. Since glutamic acid does not prevent the utilization of precursors of aspartic acid such as bicarbonate and oxalacetate which reverse cysteic acid inhibition competitively, different points of action with cysteic acid probably acting subsequent to glutamic acid are involved. The synergistic effects of combinations of the two inhibitors on L. arabinosus 17-5 offers further evidence for the. involvement of a sequence of inhibitions. REFERENCES 1. RAVEL, J. M., FELSING, B., AND SHIVE, W., J. Biol. Chem. 206, 791 (1954). 2. WOODS, L., RAVEL, J. M., AND SHIVE, W., J. Biol. Chem. 209,559 (1954). 3. RAVEL, J. M., WOODS, L., FELSING, B., AND SHIVE, W., J. Biol. Chem. 206, 391 (1954). 4. BLACK, S., AND GRAY, N. M., J. Am,. Chem. 5. BANDURSKI, R. S., AND GREINER, C. M., J. 6. VENNESLAND, B., TCHEN, T. T., AND LOEWUS, (1954).
Sot. 76, 5766 (1953). Biol. Chem. 204, 781 (1953). F. A., J. Am. Chem. Sot. 76,3358