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Synthesis of amino acids by Aerobacter aerogenes
Synthesis of Amino Acids by Aerobuctet aerogenes’ Eric B. Fowler and C. H. We&man From the Department
of Bacteriology,
Iowa State College, Ames, Iowa
Received May 13, 1953
This paper considers the conditions affecting the synthesis of glutamate, aspartate, and alanine by Aerobacter aerogenes. METHODS A. aerogenes 199 was grown in nutrient broth for 24 hr., centrifuged, washed twice with sterile saline, and diluted to a turbidity reading of 69 f 2 on a KlettSummerson photoelectric calorimeter. This suspension was used to inoculate media containing NHICI, phosphate buffer (pH = 6.8), and glucose or cr-keto acids as a carbon source. Growth was determined as turbidity after 16 hr. Other assays were conducted on the liquors after removal of cells by treatment with sulfuric acid-sodium tungstate solution (1). Residual ammonia was determined by distillation in a modified Parnas apparatus and subsequent titration with 0.005 N HCl. The amino acids were determined by microbiological assay using Leuconostoc citronorum 3081 for alanine and glutamate and Leuconostoc mesenteroides P60 for aspartate
?I.
Residual or-keto acids were determined by measurement of the evolved CO2 after oxidation with ceric sulfate at 30°C. The procedure results in recovery of about 97% of added a-ketoglutarate, pyruvate, and/or oxalacetate with a 2O-min. reaction time. Cells were grown under both aerobic and anaerobic conditions in the presence and absence of inhibitors in an attempt to correlate synthesis of amino acids and the presence of certain metabolic intermediates. Sodium arsenite was used (3) to inhibit oxidative decarboxylation of a-ketoglutarate, sodium fluoride (4) to inhibit phosphorylation, and methadone hydrochloride (5) to inhibit pyruvate oxidation.
EXPERIMENTAL RESULTS In the absence of inhibitors, anaerobiosis stimulates the formation of alanine, depresses glutamate, but has no effect on aspartate (Table I). Arsenite increases the residual cr-keto acids under both aerobiosis and 1 Supported in part by funds of the Industrial Journal Paper No. J-2323 of the Iowa Agricultural Iowa. Project No. 975. 22
Science Research Institute. Experiment Station, Ames,
AMINO
SCID
TABLE
23
SYNTHESIS
I
Effect of Anaerobiosis and Inhibitors on Amino Acid Synthesis by A. aerogenes with Glucose as Carbon Source Each flask contained 0.125% glucose, 0.15 M phosphate buffer, 0.02 M NHXl, and water to make a total volume of 8.0 ml. Time = 16 hr. Relative growth
Residual a-k& acids
= I
Amino acid concentration, .g./$ask
I-
-
Alanine
Glutamate
T
An __~~
A
An
A _~
80
176
120
120
320
120
360
272
160
136
80
400
120
128
280
64
160
80
120
120
104
253
213
184
400
384
160
120
120
Aa
An
A
An
A
None
95
23
455
24
0.003 M NaAsOs
94
22
735
0.087 M NaF
9
35
0.087 M Methadone. HCl
1
57
a A and An designate
Aspartate
aerobic and anaerobic,
All
respectively.
anaerobiosis, and under aerobic conditions there is an accompanying increase of glutamate, alanine, and aspartate. This result is to be expected if the mechanisms of formation of the precursors of the three amino acids, i.e., pyruvate, oxalacetate, and a-ketoglutarate are interdependent. The increased concentrations of alanine and aspartate indicate that in such a relationship pyruvate and oxalacetate have arisen before a-ketoglutarate, since their formation later would have been blocked by preventing dissimilation of a-ketoglutarate by arsenite. Synthesis of the three amino acids is dependent on factors other than the presence of a-keto precursors since under anaerobic conditions in hhe presence of arsenite, there results an increased concentration of residual a-keto acids but a diminution of the synthesis of both alanine and aspartate (Table I). If an inhibitor were to block the formation of a common primary precursor of interrelated compounds, in all probability there would result a diminution in the formation of all of the compounds. When sodium fluoride was used to block the aerobic formation of pyruvate, a simultaneous decrease was observed in t,he concentrations of t,he amino acids, as well as the residual a-keto acids and growth. Since fluoride interferes with phosphorylative metabolism, depression
24
ERIC
B.
FOWLER
AND
C. H.
WERKMAN
of growth and synthesis in its presence suggests the importance of a phosphorylation prior to synthesis of the three amino acids. Further, arsenite may have blocked an oxidative reaction necessary to phosphorylation, resulting in the observed accumulation of a-keto acids and a lack of their use in synthesis of amino acids. At the present stage of the investigation, we do not have an explanation of the observed increase in growth and in concentration of or-keto acids under anaerobic conditions in the presence of sodium fluoride. Methadone hydrochloride reduced the aerobic synthesis of glutamate but increased aspartate and alanine under both aerobic and anaerobic conditions. However, the organism grew in the presence of methadone only under anaerobic conditions. Elliott et al. (5) have shown an inhibition in the oxidation of glucose, lactate, and pyruvate but not of succinate by methadonerelated compounds. The data suggest that metabolite(s) from glucose accumulate and hence are more readily available to the protein synthetic mechanisms; thus increased growth results. Methadone inhibition may occur prior to the separation of aerobic from anaerobic metabolism (5). Krebs and Johnson (6) early showed a marked stimulation in synthesis of succinic acid in the presence of pyruvate under anaerobic conditions. An elucidation of the role of succinate in the synthesis of the amino acids concerned in the problem requires further study. The relatively high formation of aspartate when the system is inhibited by methadone suggests the presence of an oxalacetate-aspartate system active in the initial assimilation of ammonia, Such a system has been suggested by Virtanen and Laine (7), based on the results obtained from time experiments with expressed pea juices. The data indicate the importance of pyruvate in the synthesis of aspartate, and hence the importance of Con-fixation, perhaps by way of the Wood-Werkman reaction. Kritzman (8) has described this reaction as of primary importance in the synthesis of amino acids [cf. Fowler and Werkman (9)]. Although the results strongly suggest the reductive ammoniation of pyruvate and ammoniation of oxalacetate, such a conclusion is not valid in the absence of a transaminase block or synthesis by a purified enzyme system. The suitability of cll-keto acids as sources of carbon for growth and as precursors of the amino acids was determined (Table II). Good growth resulted when pyruvate, a-ketoglutarate, and ammonia were present. Pyruvate plus ammonia as a substrate was more efficient in promoting
AMINO
ACID
TABLE Growth
25
SYNTHESIS
II
of A. aerogenes with a-Keto Acids as Carbon Source
Each flask contained 0.15 M phosphate buffer, 0.033 M NaHC03 , and, in addition, 0.02 M NH&l, 0.02 M sodium pyruvate, and 0.01 111sodium or-ketoglutarate as noted. Water to make a total volume of 8.0 ml. Time = 16 hr.
=
=
Relative
growth
NH&+ assimilation
-
Additions
-
.Aerobic
Aerobic poles
None (buffer only) wKetoglutarate Pyruvate Pyruvate + n-ketoglutarate NH&l NH&l + cr-ketoglutarate Pyruvate + NH&l Pyruvate + NH&l + aketoglutarate
4 0 2 6 4 5 40 64
2 4 6 6 4 16
28 42
-
I
-
Anaerobic /.tmoles
-
-
-
-
-
-
-
10 49 53 93
-
42 . 66 64 92
-
growth than was a-ketoglutarate and ammonia. Pyruvate plus ammonia was more stimulatory under anaerobic conditions. The increased relative growth afforded by pyruvate (Table II) and by sodium fluoride and glucose (Table I) under anaerobic conditions suggests the importance of an unidentified metabolite in the assimilation of ammonium ion which might be a precursor of an amino acid. The metabolite would appear to arise from pyruvate on reduction; it has been shown not to be lactate. It should be noted that in some instances the ammonium ion was assimilated even though little growth was evident (Table II). W. B. Sutton (private communication) has observed active synthesis of nucleic acids with A. aerogenes prior to the attainment of a high reproduction rate. Such a synthesis would account for the utilization of ammonia in the absence of extensive growth. It is significant that marked increases in aspartate were observed whenever pyruvate served as the sole or partial source of carbon, particularly in the presence of sodium arsenite (Table III). The pyruvate-aspartate relationship has been noted (9) and explained as evidence of p-carboxylation followed by ammoniation. The results would indicate a blocking by sodium arsenite of adsorption or incorporation of the synthesized amino acid; hence, the differences in levels of aspartate (and glutamate) in the presence of the two inhibitors
26
ERIC
B.
FOWLER
AND
C. H.
TABLE
WERKMSN
III
Effect of Inhibitors on Amino Acid Acid Synthesis by A. aerogenes Growing Aerobically with ol-Keto Acids as Carbon Source Each flask contained 0.15 M phosphate buffer, 0.033 M NaHCOy , and, in addition, 0.02 M NH&I, 0.02 M sodium pyruvate, and 0.01 M sodium cr-ketoglutarate as noted. Water to make a total volume of 8.0 ml. Time = 16 hr. =
I
I:
Relative growth
Additions
None a-Ketoglutarate Pyruvate Pyruvate + a-ketoglutarate NH&l NH&l + a-ketoglutarate NH&l + pyruvate NH&l + pyruvate + a-ketoglutarate
Amino acid concentration, pg j&k 7
Alanine
3.087 M 0.0015 w
NaF
-
I_____
INaAr(ij
8 13
0.087
NaF
Aspartate
Glutamate
- _-
M 0 001.5M 0.087 u 0 0015 M 0.087 M INphaOj NaF: NaAsOx NaF
_
216 240 280 240
88 80 72 96
5 8 28 32
80
80
112 400 360
80 80 80
0.0015 ,\I NaAsO,
112 120 104 128
4 6
0 1
232 280
40 80
7 7
80 88
128 120
24 64
3 18
232 216
88 96
36 14
400 374
128 128
may be a reflection of differential incorporation. A resulting diminution in relative growth in the presence of sodium arsenite and the increased concentration of aspartate and glutamate in the growth liquors support this suggestion. This explanation does not apply in the case of alanine, however, where a higher concentration of alanine was found in the presence of sodium fluoride as compared to sodium arsenite. Present data do not explain this difference. Sodium arsenite is not as inhibitory to growth, production of the three amino acids, or accumulation of ar-keto acids from glucose, as it is when pyruvate and/or a-ketoglutarate serve as substrate (Tables I and III). These results have been interpreted as further evidence of an unidentified precursor from glucose important in ammoniation and growth. CONCLUSIONS
AND
SUMMARY
1. The action of inhibitors on the growth of Aerobacter aerogenes and on the organism’s synthesis of amino acids suggests the importance of
AMINO
ACID
SYNTHESIS
27
an unknown metabolite closely related to pyruvate and to the amino acids studied. 2. That the unknown metabolite is probably phosphorylated is deduced from the observed action of sodium fluoride. 3. The presence of an oxalacetate-aspartate system for the assimilation of ammonia is discussed. REFERENCES 1. SCHURR, D. E., THOMPSON, H. T., HENDERSON, L. M., AND ELVEHJEM, C. A., J. Biol. Chem. 182, 29-37 (1950). 2. SAUBERLICH, H. E., AND BAUMANN, C. H., J. Biol. Chem. 177, 545-51 (1949). 3. KREBS, H. A., AND JOHNSON, W. A., Enzymologia 4, 148 (1937). 4. UTTER, M. F., AND WERKMAN, C. H., J. Biol. Chem. 148, 289-300 (1942). 5. ELLIOTT, H. W., WARRENS, A. E., AND JAMES, H. P., J. Phurmacol. Exptl. Therap. 91, 98-102 (1947). 6. KREBS, H. A., AND JOHNSON, W. .4., Biochem. J. 31, 645-660 (1937). 7. VIRTANEN, A. I., AND LAINE, T., Nature 141, 748 (1938). 8. KRITZMAN, M. G., Biokhimiya 9, 379-88 (1944). 9. FOWLER, E. B., AND WERKMAN, C. H., Arch. Biochem. and Biophys. 36, 365-70 (1952).
of Bacteriology,
Iowa State College, Ames, Iowa
Received May 13, 1953
This paper considers the conditions affecting the synthesis of glutamate, aspartate, and alanine by Aerobacter aerogenes. METHODS A. aerogenes 199 was grown in nutrient broth for 24 hr., centrifuged, washed twice with sterile saline, and diluted to a turbidity reading of 69 f 2 on a KlettSummerson photoelectric calorimeter. This suspension was used to inoculate media containing NHICI, phosphate buffer (pH = 6.8), and glucose or cr-keto acids as a carbon source. Growth was determined as turbidity after 16 hr. Other assays were conducted on the liquors after removal of cells by treatment with sulfuric acid-sodium tungstate solution (1). Residual ammonia was determined by distillation in a modified Parnas apparatus and subsequent titration with 0.005 N HCl. The amino acids were determined by microbiological assay using Leuconostoc citronorum 3081 for alanine and glutamate and Leuconostoc mesenteroides P60 for aspartate
?I.
Residual or-keto acids were determined by measurement of the evolved CO2 after oxidation with ceric sulfate at 30°C. The procedure results in recovery of about 97% of added a-ketoglutarate, pyruvate, and/or oxalacetate with a 2O-min. reaction time. Cells were grown under both aerobic and anaerobic conditions in the presence and absence of inhibitors in an attempt to correlate synthesis of amino acids and the presence of certain metabolic intermediates. Sodium arsenite was used (3) to inhibit oxidative decarboxylation of a-ketoglutarate, sodium fluoride (4) to inhibit phosphorylation, and methadone hydrochloride (5) to inhibit pyruvate oxidation.
EXPERIMENTAL RESULTS In the absence of inhibitors, anaerobiosis stimulates the formation of alanine, depresses glutamate, but has no effect on aspartate (Table I). Arsenite increases the residual cr-keto acids under both aerobiosis and 1 Supported in part by funds of the Industrial Journal Paper No. J-2323 of the Iowa Agricultural Iowa. Project No. 975. 22
Science Research Institute. Experiment Station, Ames,
AMINO
SCID
TABLE
23
SYNTHESIS
I
Effect of Anaerobiosis and Inhibitors on Amino Acid Synthesis by A. aerogenes with Glucose as Carbon Source Each flask contained 0.125% glucose, 0.15 M phosphate buffer, 0.02 M NHXl, and water to make a total volume of 8.0 ml. Time = 16 hr. Relative growth
Residual a-k& acids
= I
Amino acid concentration, .g./$ask
I-
-
Alanine
Glutamate
T
An __~~
A
An
A _~
80
176
120
120
320
120
360
272
160
136
80
400
120
128
280
64
160
80
120
120
104
253
213
184
400
384
160
120
120
Aa
An
A
An
A
None
95
23
455
24
0.003 M NaAsOs
94
22
735
0.087 M NaF
9
35
0.087 M Methadone. HCl
1
57
a A and An designate
Aspartate
aerobic and anaerobic,
All
respectively.
anaerobiosis, and under aerobic conditions there is an accompanying increase of glutamate, alanine, and aspartate. This result is to be expected if the mechanisms of formation of the precursors of the three amino acids, i.e., pyruvate, oxalacetate, and a-ketoglutarate are interdependent. The increased concentrations of alanine and aspartate indicate that in such a relationship pyruvate and oxalacetate have arisen before a-ketoglutarate, since their formation later would have been blocked by preventing dissimilation of a-ketoglutarate by arsenite. Synthesis of the three amino acids is dependent on factors other than the presence of a-keto precursors since under anaerobic conditions in hhe presence of arsenite, there results an increased concentration of residual a-keto acids but a diminution of the synthesis of both alanine and aspartate (Table I). If an inhibitor were to block the formation of a common primary precursor of interrelated compounds, in all probability there would result a diminution in the formation of all of the compounds. When sodium fluoride was used to block the aerobic formation of pyruvate, a simultaneous decrease was observed in t,he concentrations of t,he amino acids, as well as the residual a-keto acids and growth. Since fluoride interferes with phosphorylative metabolism, depression
24
ERIC
B.
FOWLER
AND
C. H.
WERKMAN
of growth and synthesis in its presence suggests the importance of a phosphorylation prior to synthesis of the three amino acids. Further, arsenite may have blocked an oxidative reaction necessary to phosphorylation, resulting in the observed accumulation of a-keto acids and a lack of their use in synthesis of amino acids. At the present stage of the investigation, we do not have an explanation of the observed increase in growth and in concentration of or-keto acids under anaerobic conditions in the presence of sodium fluoride. Methadone hydrochloride reduced the aerobic synthesis of glutamate but increased aspartate and alanine under both aerobic and anaerobic conditions. However, the organism grew in the presence of methadone only under anaerobic conditions. Elliott et al. (5) have shown an inhibition in the oxidation of glucose, lactate, and pyruvate but not of succinate by methadonerelated compounds. The data suggest that metabolite(s) from glucose accumulate and hence are more readily available to the protein synthetic mechanisms; thus increased growth results. Methadone inhibition may occur prior to the separation of aerobic from anaerobic metabolism (5). Krebs and Johnson (6) early showed a marked stimulation in synthesis of succinic acid in the presence of pyruvate under anaerobic conditions. An elucidation of the role of succinate in the synthesis of the amino acids concerned in the problem requires further study. The relatively high formation of aspartate when the system is inhibited by methadone suggests the presence of an oxalacetate-aspartate system active in the initial assimilation of ammonia, Such a system has been suggested by Virtanen and Laine (7), based on the results obtained from time experiments with expressed pea juices. The data indicate the importance of pyruvate in the synthesis of aspartate, and hence the importance of Con-fixation, perhaps by way of the Wood-Werkman reaction. Kritzman (8) has described this reaction as of primary importance in the synthesis of amino acids [cf. Fowler and Werkman (9)]. Although the results strongly suggest the reductive ammoniation of pyruvate and ammoniation of oxalacetate, such a conclusion is not valid in the absence of a transaminase block or synthesis by a purified enzyme system. The suitability of cll-keto acids as sources of carbon for growth and as precursors of the amino acids was determined (Table II). Good growth resulted when pyruvate, a-ketoglutarate, and ammonia were present. Pyruvate plus ammonia as a substrate was more efficient in promoting
AMINO
ACID
TABLE Growth
25
SYNTHESIS
II
of A. aerogenes with a-Keto Acids as Carbon Source
Each flask contained 0.15 M phosphate buffer, 0.033 M NaHC03 , and, in addition, 0.02 M NH&l, 0.02 M sodium pyruvate, and 0.01 111sodium or-ketoglutarate as noted. Water to make a total volume of 8.0 ml. Time = 16 hr.
=
=
Relative
growth
NH&+ assimilation
-
Additions
-
.Aerobic
Aerobic poles
None (buffer only) wKetoglutarate Pyruvate Pyruvate + n-ketoglutarate NH&l NH&l + cr-ketoglutarate Pyruvate + NH&l Pyruvate + NH&l + aketoglutarate
4 0 2 6 4 5 40 64
2 4 6 6 4 16
28 42
-
I
-
Anaerobic /.tmoles
-
-
-
-
-
-
-
10 49 53 93
-
42 . 66 64 92
-
growth than was a-ketoglutarate and ammonia. Pyruvate plus ammonia was more stimulatory under anaerobic conditions. The increased relative growth afforded by pyruvate (Table II) and by sodium fluoride and glucose (Table I) under anaerobic conditions suggests the importance of an unidentified metabolite in the assimilation of ammonium ion which might be a precursor of an amino acid. The metabolite would appear to arise from pyruvate on reduction; it has been shown not to be lactate. It should be noted that in some instances the ammonium ion was assimilated even though little growth was evident (Table II). W. B. Sutton (private communication) has observed active synthesis of nucleic acids with A. aerogenes prior to the attainment of a high reproduction rate. Such a synthesis would account for the utilization of ammonia in the absence of extensive growth. It is significant that marked increases in aspartate were observed whenever pyruvate served as the sole or partial source of carbon, particularly in the presence of sodium arsenite (Table III). The pyruvate-aspartate relationship has been noted (9) and explained as evidence of p-carboxylation followed by ammoniation. The results would indicate a blocking by sodium arsenite of adsorption or incorporation of the synthesized amino acid; hence, the differences in levels of aspartate (and glutamate) in the presence of the two inhibitors
26
ERIC
B.
FOWLER
AND
C. H.
TABLE
WERKMSN
III
Effect of Inhibitors on Amino Acid Acid Synthesis by A. aerogenes Growing Aerobically with ol-Keto Acids as Carbon Source Each flask contained 0.15 M phosphate buffer, 0.033 M NaHCOy , and, in addition, 0.02 M NH&I, 0.02 M sodium pyruvate, and 0.01 M sodium cr-ketoglutarate as noted. Water to make a total volume of 8.0 ml. Time = 16 hr. =
I
I:
Relative growth
Additions
None a-Ketoglutarate Pyruvate Pyruvate + a-ketoglutarate NH&l NH&l + a-ketoglutarate NH&l + pyruvate NH&l + pyruvate + a-ketoglutarate
Amino acid concentration, pg j&k 7
Alanine
3.087 M 0.0015 w
NaF
-
I_____
INaAr(ij
8 13
0.087
NaF
Aspartate
Glutamate
- _-
M 0 001.5M 0.087 u 0 0015 M 0.087 M INphaOj NaF: NaAsOx NaF
_
216 240 280 240
88 80 72 96
5 8 28 32
80
80
112 400 360
80 80 80
0.0015 ,\I NaAsO,
112 120 104 128
4 6
0 1
232 280
40 80
7 7
80 88
128 120
24 64
3 18
232 216
88 96
36 14
400 374
128 128
may be a reflection of differential incorporation. A resulting diminution in relative growth in the presence of sodium arsenite and the increased concentration of aspartate and glutamate in the growth liquors support this suggestion. This explanation does not apply in the case of alanine, however, where a higher concentration of alanine was found in the presence of sodium fluoride as compared to sodium arsenite. Present data do not explain this difference. Sodium arsenite is not as inhibitory to growth, production of the three amino acids, or accumulation of ar-keto acids from glucose, as it is when pyruvate and/or a-ketoglutarate serve as substrate (Tables I and III). These results have been interpreted as further evidence of an unidentified precursor from glucose important in ammoniation and growth. CONCLUSIONS
AND
SUMMARY
1. The action of inhibitors on the growth of Aerobacter aerogenes and on the organism’s synthesis of amino acids suggests the importance of
AMINO
ACID
SYNTHESIS
27
an unknown metabolite closely related to pyruvate and to the amino acids studied. 2. That the unknown metabolite is probably phosphorylated is deduced from the observed action of sodium fluoride. 3. The presence of an oxalacetate-aspartate system for the assimilation of ammonia is discussed. REFERENCES 1. SCHURR, D. E., THOMPSON, H. T., HENDERSON, L. M., AND ELVEHJEM, C. A., J. Biol. Chem. 182, 29-37 (1950). 2. SAUBERLICH, H. E., AND BAUMANN, C. H., J. Biol. Chem. 177, 545-51 (1949). 3. KREBS, H. A., AND JOHNSON, W. A., Enzymologia 4, 148 (1937). 4. UTTER, M. F., AND WERKMAN, C. H., J. Biol. Chem. 148, 289-300 (1942). 5. ELLIOTT, H. W., WARRENS, A. E., AND JAMES, H. P., J. Phurmacol. Exptl. Therap. 91, 98-102 (1947). 6. KREBS, H. A., AND JOHNSON, W. .4., Biochem. J. 31, 645-660 (1937). 7. VIRTANEN, A. I., AND LAINE, T., Nature 141, 748 (1938). 8. KRITZMAN, M. G., Biokhimiya 9, 379-88 (1944). 9. FOWLER, E. B., AND WERKMAN, C. H., Arch. Biochem. and Biophys. 36, 365-70 (1952).