BIOCHEMICAL TRANSFORMATIONS AS DETERMINED BY COMPETITIVE ANALOGUE-METABOLITE GROWTH INHIBITIONS

BIOCHEMICAL TRANSFORMATIONS AS DETERMINED BY COMPETITIVE ANALOGUE-METABOLITE GROWTH INHIBITIONS IV. PREVENTION OF PANTOTHENIC ACID SYNTHESIS BY CYSTEIC ACID* BY JOANNE MACOW RAVEL AND WILLIAM SHIVE (From the Clayton Biochemical Institute and the Department of chemistry, The University of Texas, Austin) (Received for publication, August 13,1946) The study of the metabolism of cells by a method termed inhibition analysis (l-3) led to the initial proposal that the antibacterial index (4) is a function of the inhibited enzyme system which becomes the limiting factor for growth of the organism. It was further proposed that if the product of this enzyme system is supplied to the organism the analogue either becomes ineffective as a growth inhibitor or at higher concentrations affects another enzyme system. In the latter case a higher antibacterial index, corresponding to this second enzyme system, is obtained. Precursors of the metabolite may be effective in preventing inhibition of growth by the analogue over a range of concentrations, depending upon the effectiveness with which they are transformed into the metabolite. In the present investigation paralleling recent work on the inhibition of growth of Escherichia coli with hydroxyaspartic acid (l), cysteic acid was found to inhibit growth of that organism but only in the absence of p-alanine or pantothenic acid. The inhibition was competitively prevented by aspartic acid; hence, because of the structural similarity of cysteic acid and aspartic acid, it appears that the enzyme system converting aspartic acid to fl-alanine was blocked by the inhibitor. An unusual result was obtained with glutamic acid which was found in many tests to be more effective than aspartic acid in preventing the toxicity of cysteic acid. In order to investigate further what appears to be greater activity of a precursor with respect to the metabolite, tests determining the comparative effects of aspartic acid, glutamic acid, a-ketoglutaric acid, and mixtures of cu-ketoglutaric acid and aspartic acid were carried out simultaneously. Cysteic acid also inhibits the growth of Lactobacillus casei, Lactobaeillus arabinosus 17-5, and Leuconostoc mesenteroides P-6, and in all cases the inhibition is reversed competitively by aspartic acid but not by glutamic *For the most part from a thesis submitted by Joanne Macow Ravel to The University of Texas in partial fulfilment of the degree of Master of Arts, February, 1946. 47

48 BIOCHEMICAL TRANSFORMATIONS. IV acid. Details of these experiments and their implications are presented below. EXPERIMENTAL Materials-a-Ketoglutaric acid was prepared by a combination of the methods of Blaise and Gault (5) and Gabriel (6). TABLE I E$ects of Aspartic Acid, &Alanine, and Pantothenic Acid on Toxicity of Cysteic Acid for Escherichia coli Incubated 16 hours at 37-38. I-Aspartic acid --. - CCysteic acid y gcr IO cc. 1 3 3 l,ooo l,@jo 3,6 3,6 1, 3,6 3, Antibacterial Revkrsing agent y per 1 cc. 3 3 3 1 1 1 3 3 3 1 3 index, 3 ca. - I S.lvanomrter - 52. 53.5 5.5 5. 52. 2.5 54. 51. 2. 54. 49. 1. 1. 2. Pantothenic acid 1. 55. 1,6 1. 53. 3, 1. 14. 1, 1. 2. &Alanine 1 53. 1, 1 53. 3, 1 22. 1,6 1 6. readings* Antibacterial index, >3 in presence of &alanine or pantothenic acid * A measure of culture turbidity; distilled water reads, an opaque object 1. dl- para -Hydroxyaspartic acid was prepared by the method of Dakin (7). Only this isomer was used in the tests described in this investigation. I-Cysteic acid monohydrate was prepared from l-cystine by the method described by Clarke (8).

J. M. RAVEL AN D W. BHIVE 49 Testing Method-Tests with Escherichia wli were performed as previously described (1). The casein hydrolyzed with trypsin was omitted from the basal medium. For lactic acid bacteria, a previously described procedure was used (9) with a basal medium containing no aspartic acid. Time and temperature of incubation are given in Tables I to IV. Results-The results as shown in Table I indicate that cysteic acid is toxic to Escherichia coli and that the toxicity is prevented competitively by aspartic acid, the antibacterial index being approximately 3. The antibacterial index varied from one test to another but was usually in the range of 3 to 1. However, no inhibition of growth was obtained in a medium containing 1 y of pantothenic acid or 1 y of @-alanine unti! irreversible toxicity with respect to aspartic acid, pantothenic acid, or /3-alanine was attained at levels of cysteic acid varying from 3 to 3 mg. per 1 cc. The inhibition of growth of Lactobacillus casei and Lactobacillus arabinosus 17-5 by cysteic acid is shown in Table II. The inhibition is prevented competitively in both cases by aspartic acid. The antibacterial index is approximately 3 with either organism. Similar tests with Leuconostoc mesenteroides P-6 gave analogous results with an antibacterial index of about 1. P-Alanine had no effect on the toxicity of cysteic acid for Lactobacillus arabinosus, the only species of lactic acid bacteria so tested. The comparative effects of aspartic acid, glutamic acid, a-ketoglutaric acid, and a mixture of aspartic acid and cy-ketoglutaric acid on the toxicity of cysteic acid and hydroxyaspartic acid for Escherichia wli are shown in Tables III and IV. In order to standardize the results, simultaneous tests inoculated from the same culture of the organism were repeated several times. Some generalizations can be made concerning the results of these tests. In regard to inhibition of growth with cysteic acid, the following comparisons can be made with regard to the effectiveness of these materials in reversing the toxicity: (1) Aspartic acid prevented the inhibition competitively, the antibacterial index being approximately 3 and sometimes near 1. (2) Glutamic acid was about 3 times as effective as aspartic acid in preventing the inhibition, but often showed some decrease in activity at higher concentrations, 3 y per 1 cc. (3) cr-ketoglutaric acid was approximately as effective as aspartic acid in reversing the inhibition; however, at concentrations of 1 to 3 y per 1 cc. the keto acid appeared to become progressively less effective. (4) Mixtures of ar-ketoglutaric acid and aspartic acid were equally, if not more, effective than amounts of glutamic acid from which such mixtures could be derived by transamination. Regarding inhibition of growth of Escherichia coli with hydroxyaspartic acid, the following generalizations

41 BIOCHEMICAL TRANSFORMATIONS. IV can be made: (1) Aspartic acid reversed the inhibition competitively, the antibacterial index being 1 to 3. (2) Glutamic acid was approximately as effective as aspartic acid, except at higher concentrations, TABLE Growth Inhibition by Cysteic Acid and Its Reversal by Aspartic Acid II I-Cyst&c acid I-Aspartic acid 7 per 5 CC. -f per 5 cc. 1 1 1 3 1 l,c@o 1 3,ooo 1 3 3 3 l,@)o 3 3,~ 3 1,6 3 1 l,w 1 3,6 1 1, 19 3, 1 3 3,ooo 3 1, 3 3,6 3 1,ooo 3 3, 3 6 f3,oc o 6 2, 6 6,9 6 2, 6 1, 1, 1, 3, Galvanometer readings Loclobacillus casei aclobacillus arabinosust 13.6 37.5 2. 18. 15.2 5. 56.2 22.8 17. 6. 4. 6. 47. 29.8 19. 9. 61. 59. 62. 32. 14.5 8. 62.2 62.2 6.5 31.5 7.6 36.5 47.5 32. 52. 37. 26.2 9.5 5. 57. 59. 4.6 16. 8.2 69.7 67. 47. 26. 11.2 6. 76. 7.2 57. 29.5 6.2 7.2 7. Antibacterial index.. 3 Ca. 3 Ca. * Incubated 3 days at 37-38. t Incubated 18 hours at 3. 1 to 3 y per 1 cc., at which the relative activity of glutamic acid steadily decreased with increases in concentration. (3) a-ketoglutaric acid was approximately as active as aspartic acid only at very low con-

J. M. RAVEL AND W. SElVE 411 TABLE Comparative Eflects of Aspartic Acid, Glzttamic Acid, and a-ketoglutaric Acid on Toxicity of Cysteic Acid for Escherichia coli Incubated 16 hours at 37-38. III [-Cysteic acid 7 per 1 cc. &Aspartic 44. 3 46. 1 7. 3 3. 1 3 l,ooo 3, 1, 3 l,c@o 3, 1, acid Galvanometer readings I-Glutmic acid a-ketoglutaric acid* I-Aspartic acid and a-ketoglutaric acid y per 1 cc. 1 y per 1 cc.? 45. 4. 19. 9. 3. 3 y per 1 cc.? 45.5 42. 26.5 13. 3. 1 y per 1 cc.t 44. 43. 1. 6. 47. 37. 8. 3. 2. 44. 44. 41.5 41. 6. 41. 5. 14.5 4.5 44.5 46. 44. 47. l,ooo 42. 44.8 41. 39. 3, 6. 42.5 18. 4. 1, 2. 7. 4. 14. 3, 3. 5. 3 y per 1 cc.t 45. 49. 47. 45. 3, 44.2 45. 23. 44.8 1,9 3.5 31. 4. 43.2 3,9 4. 4.8 Antibacterial index, 3 Cu. * Autoclaved separately and added aseptically to the sterilized medium. t For the mixture of I-aspartic and a-ketoglutaric acids, the indicated weight of each was added.

412 BIOCHEMICAL TBANSFORMATIONS. IV TABLE IV Comparative Effects of Aspartic Acid, Glutamic Acid, and a-ketoglutaric Acid on Toxicity of Hydroxyaspartic Acid for Escherichia coli Incubated 16 hours at 37-38. dl-#ima- HydroayiTpartic I-Aspartic y per 1 cc. 43.5 3 42. 1 9.8 3 1.5 acid Galvanometer readings I-Aspartic acid and I-Glutamic acid a-ketoglutaric acid* wketoglutaric acid* y per 1 cc. 1 y per 1 cc.t 44. 44. 44.3 44. 3 43. 45. 44. 45. 1 35. 42.4 25.5 42. 3 6. 5. 3.2 11. 1 2. 1.2 2. 2. 3 y per 1 cc.t 46. 47.8 48. 46. loo 42.5 41. 42. 43. 3 15. 1. 8. 21. 1 3. 1.5 2. 9. 3 1.5 1.5 2.2 2.7 1 y per 1 cc.t 44. 45. 47. 47.5 1 43. 43.5 44. 44. 3 27. 31. 14.5 31. 1 13. 2. 2. 19. 3 3.7 1.5 2. 12. 3 y per 1 cc.t 45. 47.7 47. 47. 1 43.5 47.5 44. 43.5 3 4. 45. 18. 42. 1 24. 2. 2. 3.5 39 19. 1.5 3. 29. Antibacterial index, 1-3 * Autoclaved separately and added aseptically to the medium. t For the mixture of Z-aspartic and cu-ketoglutaric acids, the indicated weight of each was added.

J. M. RAVEL AND W. SHIV3 413 centrations, 1 y per 1 cc., and further increases in the concentration of the keto acid had little effect on the toxicity of hydroxyaspartic acid. (4) Mixtures of ar-ketoglutaric acid and aspartic acid were little if any more active than the amount of aspartic acid in the mixture. Separate experiments indicated that several other metabolites are somewhat effective in reversing the toxicity of cysteic acid for Escherichia co%. Among these were thiamine, leucine, asparagine, arginine, proline, hydroxyproline, isoleucine, and valine. DISCUSSION The competitive cysteic acid-aspartic acid growth inhibition obtained with Escherichia coli appears to be a function of the enzyme system which decarboxylates aspartic acid to /I-alanine. As no other enzyme system utilizing aspartic acid is blocked by cysteic acid, the addition of @-alanine or pantothenic acid to the medium supplies the limiting factor for growth; hence, cysteic acid is then no longer toxic at any level up to 3 mg. per 1 cc., at which level it is irreversibly toxic with respect to aspartic acid, p-alanine, or pantothenic acid. In other words, cysteic acid prevents only one necessary reaction of aspartic acid, its decarboxylation to /!?- alanine, in Escherichia coli. It has not been determined whether cysteic acid is decarboxylated to taurine by the enzyme and merely occupies the enzyme during the reaction or whether cysteic acid forms a complex with the enzyme which does not react further. The inhibition of growth of Lactobacillus casei, Lactobacillus arabinosus, and Leuconostoc mesenteroides with cysteic acid is competitive in nature; that is, aspartic acid and cysteic acid appear to compete for a common enzyme which performs some function of aspartic acid. This function is not /3-alanine synthesis in the case of Lactobacillus arabinosus. The enhanced effect of glutamic acid over aspartic acid in reversing cysteic acid toxicity for Escherichia coli is indeed an unusual effect, that of a precursor of a metabolite being more active than the metabolite in reversing an inhibition. Two explanations for this effect have been considered. The first is that cr-ketoglutaric acid is converted into glutamic acid by an enzyme system of which the keto acid is the limiting factor. As Escherichia coli is known to contain transaminase (lo), the conversion of the glutamic acid to aspartic acid and more a-ketoglutaric acid would take place rapidly. The a-ketoglutaric acid could then be used again, the limiting factors for this cycle being the rates of the reactions and the utilization of glutamic acid and a-ketoglutaric acid for other purposes in the organism. By such a process, glutamic acid could show a greater activity than aspartic acid. The second explanation is that cr-ketoglutaric acid or some closely related compound may increase the enzyme concentration of the svstem which decarboxvlates asnartic acid.

414 BIOCHEMICAL TRANSFORMATIONS. IV A study of the comparative effects of aspartic acid, glutamic acid, and cu-ketoglutaric acid on the toxicity of cysteic acid and hydroxyaspartic acid for Escherichia coli was made as indicated in Tables III and IV in order to determine which, if either, of these two explanations might be correct. A mixture of a-ketoglutaric acid and aspartic acid is equally, if not more, effective than glutamic acid in preventing the toxicity of cysteic acid. Hence, an initial transamination reaction which is known to be rapid for enzyme preparations from Escherichia wli (1) is indicated. The effect of glutamic acid, a-ketoglutaric acid, and a mixture of cr-ketoglutaric acid and aspartic acid on the toxicity of hydroxyaspartic acid shows that the enhanced effect of glutamic acid over aspartic acid does not exist with this inhibitor. If an increased production of enzyme had resulted, the effect of increased activity of the glutamic acid should be common to both inhibitors since they apparently prevent the functioning of the same enzyme under the testing conditions (1). It might be expected that precursors of aspartic acid should act similarly in preventing the toxicity of each inhibitor. However, the relatively high activity of (Yketoglutaric acid in reversing the toxicity of cysteic acid and the low activity of that keto acid in reversing the toxicity of hydroxyaspartic acid appear to result from the variation in the ability of the organism to transform a-ketoglutaric acid into glutamic acid in the presence of the inhibitors. Such a variation in activity could be explained either by hydroxyaspartic acid directly or indirectly inhibiting this transformation or by cysteic acid aiding the conversion. Cysteic acid and a-ketoglutaric acid have been shown to undergo transamination in the presence of some enzyme preparations (11). Further study is being given this aspect of the problem. Grateful acknowledgment is made to Dr. Beverly M. Guirard for valuable help with much of the testing. SUMMARY The toxicity of cysteic acid for Lactobacillus arabinosus, Lactobacillus casei, Leuconostoc mesenteroides, and Escherichia coli is prevented competitively by aspartic acid, the antibacterial indices being approximately 3, 3, 1, and 3, respectively. Cysteic acid prevents the synthesis of pantothenic acid in Escherichia coli by blocking an enzyme of the system which decarboxylates aspartic acid to form /3-alanine. Either &ala&e or pantothenic acid completely prevents the toxicity of cysteic acid up to levels of 3 mg. of inhibitor per 1 cc. At this level, cysteic acid is irreversibly toxic with respect to aspartic acid, p-alanine, or pantothenic acid. Glutamic acid is about 3 times as effective as aspartic acid in preventing

J. M. RAVEL AND W. SHITE 415 the toxicity of cysteic acid for Escherichia coli. A study of the comparative effects of aspartic acid, glutamic acid, ar-ketoglutaric acid, and mixtures of ar-ketoglutaric acid and aspartic acid on the toxicity of cysteic acid and hydroxyaspartic acid is reported. Some explanations of the enhanced effect of glutamic acid over aspartic acid on the toxicity of cysteic acid are discussed. BIBLIOGRAPHY 1. Shive, W., and Macow, J., J. Biol. Chem., 162,451 (1946). 2. Shive, W., and Roberts, E. C., J. Biol. Chem., 162, 463 (1946). 3. Beerstecher, E., Jr., and Shive, W., J. Biol. Chem., 164, 53 (1946). 4. McIlwain, H., Brit. J. Erp. Path., 23, 95 (1942). 5. Blaise, E. E., and Gault, H., Compt. rend. Acad., 147, 198 (198). 6. Gabriel, S., Ber. them. Ges., 42, 655 (199). 7. Dakin, H. D., J. Biol. Chem., 48, 273 (1921). 8. Clarke, H. T., in Organic syntheses, New York, 29,23 (1939). 9. McMahan, J. R., and Snell, E. E., J. Biol. Chem., 162, 83 (1944). 1. Lichstein, H. C., and Cohen, P. P., J. BioZ. Chem., 167, 85 (1945). 11. Cohen, P. P., J. BioZ. Chem., 136, 565 (194).

BIOCHEMICAL TRANSFORMATIONS AS DETERMINED BY COMPETITIVE ANALOGUE-METABOLITE GROWTH INHIBITIONS: IV. PREVENTION OF PANTOTHENIC ACID SYNTHESIS BY CYSTEIC ACID Joanne Macow Ravel and William Shive J. Biol. Chem. 1946, 166:47-415. Access the most updated version of this article at http://www.jbc.org/content/166/2/47.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites references, of which can be accessed free at http://www.jbc.org/content/166/2/47.citation.full.h tml#ref-list-1