AMINO ACID BIOSYNTHESIS IN ESCHERICHIA COLT: ISOTOPIC COMPETITION WITH C 4-GLUCOSE

Transcription

1 AMINO ACID BIOSYNTHESIS IN ESCHERICHIA COLT: ISOTOPIC COMPETITION WITH C 4-GLUCOSE BY PHILIP H. ABELSON* (From the Department of Terrestrial Magnetism, Carnegie Institution of Washington, Washington, D. C.) (Received for publication, July 27, 1953) The isotopic competition method has been applied in this laboratory to a number of problems in microbial metabolism (l-4). Thus, it was found that certain exogenous unlabeled amino acids depress the incorporation of exogenous C14, supplied as C1402, into the protein of Escherichia coli (3). The results obtained therefore provided a measure of the relative utilization of competing exogenous and endogenous amino acids. From the observed preferential utilization of some exogenous amino acids, several metabolic relationships were deduced. These studies have now been extended to isotopic competition experiments with C 4-glucose. The use of this radioactive compound instead of Cl402 has considerably increased the scope of this method. The present paper is concerned with the biosynthesis of a number of amino acids as well as with some features of the Krebs cycle in E. coli. Methods Cultural Methods--The general methods used have been previously described (3, 5). For the competition experiments a culture of E. coli, strain B, growing exponentially at 37 in unlabeled glucose-salt medium, was centrifuged, and the bacteria were resuspended in fresh glucose-free salt medium to give an optical density of at 650 rnp. 20 ml. aliquot cultures were placed in 500 ml. plastic bottles and randomly C14-labeled glucose (10 PC., 4 mg. per bottle) and any of the desired unlabeled competitors (0.1 to 0.2 mg. per ml.) were added. The bottles were stoppered and incubated with shaking at 37 until the optical density approximately doubled (1 hour). Fractionation-The organisms were harvested and extracted successively with cold trichloroacetic acid, alcohol, alcohol-ether, and hot trichloroacetic acid (5). The resulting protein residues (about, 1 mg. from each bottle) were hydrolyzed with 6 N hydrochloric acid at 110 for 15 hours. Excess hydrochloric acid was then removed by evaporation. Chromatograms and Radioautographs-Each hydrolysis residue was dis- * Nom at, Geophysical I,nhor:Ltory, Cnrnegic Institjutjion of Washington, Washington, D. C. 335

2 336 AMINO ACID IN E. COLI solved in water and transferred to Whatman No. 1 paper for two-dimensional chromatography with secondary butanol-formic acid-water (70: lo:- 20), followed by phenol-water-28 per cent ammonia (SO: 20: 1). The resulting chromatograms were dried and radioautographed. The radioautographs revealed the location of the various amino acids; the chromatographic patterns obtained resembled a previously illustrated one (6). The radioactivity of the amino acids was determined in situ with a thin end window counter. A number of hydrolysis artifacts were noted, especially in the case of the aromatic and sulfur-containing amino acids and histidine. Also, the chromatographic technique did not give complete resolution of all the amino acids. These shortcomings necessitated special procedures in some cases. Cysteine and methionine were studied as cysteic acid and methionine sulfoxide after oxidation with hydrogen peroxide. In this manner a separation of methionine from valine was achieved. The unresolved amino acids, leucine, isoleucine, and phenylalanine, were studied either after resolution by separate chromatographic techniques or by an application of the isotopic competition method. It was found that effects on the radioactivity of any member of this trio could be examined by culturing the bacteria in the presence of the respective unlabeled complementary pair, thus suppressing all radioactivity in these complementary amino acids; this procedure was validated by appropriate control experiments. Determination of Competition-The content of relevant amino acids in the protein residue was determined in experiments by using CY4-glucose without addition of amino acids. Under these conditions the radioactivity of each amino acid furnishes a measure of its relative content in the protein. The results agreed well with values obtained by elution of the amino acids and determination by standard methods. The purity of the radioactive amino acids was checked by eluting, rechromatographing with t,he respective authentic carriers, and comparing radioautographs with ninhydrin spots on paper. Under the conditions employed, the amino acid composition of the bacterial protein residue was found approximately constant. The competitive effects of various supplements were studied in similar experiments in which the bacteria were cultured in the presence of the desired supplements. The effects obtained are expressed as per cent radioactivity of a given bacterial amino acid relative to control, i.e. as counts per minute of the amino acid per unit of protein from the competition experiment divided by counts per minute per unit of protein from the control experiment (without added amino acids) multiplied by. Compounds Used-The randomly labeled C14-glucose was prepared from canna leaves (7, 8) with BaC1403 (obtained from the Oak Ridge National Laboratory) as a source of CY402. The L-amino acids used were commercial products. Several of the keto acids including d-cr-keto$-methylvaleric acid (the keto analogue of L-isoleucine) were kindly furnished by Dr. Alton

3 P. H. ARELSON 337 Meister. Synthetic N -acetyl-n-ornithine and nn-glutamic r-semialdehyde were prepared by Dr. Henry J. Vogel. RESULTS AND DISCUSSION In the present, experiments the added unlabeled carbon compounds were found either to be inactive as competitors with CY4-glucose or to affect the radioactivity of a single amino acid or that of a group of amino acids. Families of metabolically related amino acids were thus discernible. The interpretation of isotopic competition results has been considered previously (3). As discussed below, a finding of partial or complete competition by an added substance with CY4-glucose in the formation of a given biosynthetic product is taken as an indication that the added substance is a normal precursor of this product; however, this method, like others, may not differentiate between a normal precursor and a substance which is readily transformable into a normal precursor or the final product. On the other hand, failure to obtain competition in a given pathway does not necessarily indicate that the substance tested is not an intermediate in this pathway. Glutamic Family-Evidence has previously been obtained that in E. cobi glutamic acid gives rise to arginine via ornithine and citrulline (3). Ornithine was recently shown to be formed from glutamic acid via N-acetylglutamic acid, N-acetylglutamic r-semialdehyde, and N -acetylornithine (9, 10, 4). Glutamic acid has also been reported to be metabolically related to proline in this species (11, 12), and glutamic r-semialdehyde, which is in equilibrium with the cyclized A -pyrroline-5-carboxylic acid, has been shown to be an intermediate between glutamic acid and proline (13), but not between glutamic acid and ornithine (10, 4). Competition experiments, as presented in Table I, are not only in qualitative agreement with the previous findings but also provide evidence that glutamic acid and glutamic y- semialdehyde (4) are quantitatively major precursors of proline, and similarly that glutamic acid, Na-acetylornithine (4), ornithine, and citrulline are major precursors of arginine. The somewhat higher residual radioactivity of protein arginine obtained with glutamic acid, ornithine, or Nclacetylornithine as competitor, compared to that obtained with citrulline or arginine as competitor, is probably due to incorporation of C4, derived from the C14-glucose via C1402, into the amidine carbon of arginine (3). These results thus support the accompanying pathways. 7 Glutamic acid \ glutamic 7.semialdehyde -+ Al-pyrroline-5-carboxylic acid * proline N-acetylglutamic --f N-acetylglutamic r-semialdehyde -+ iv -acetylacid ornithine + ornithine -+ citrulline --+ arginine

4 338.\MINO ACID IS E. COT,1 Aspartic Family-Competition studies with aspartic acid revealed that addition of this amino acid diminishes the incorporation of CL4 into the aspartic acid, threonine, isoleucine, methionine, and lysine of the bacterial TABLE Metabolism of C14-Glucose by E. coli; Effect of Supplements on Radioactivity of Amino Acids in Protein Residue (As Per Cent Radioactivity o,f Protein Amino Acids Relative to Control)* Supplement I (;lu Arg Pro Asp Thr Iso 1 vkt Lys Ala Vs ;eu None (control).... Glutamate.... N -Acetylornithine... Omithine... Citrulline... Arginine... Glutamic 7.semialdehyde... Proline... Aspartate... Homoserine... Threonine... a-ketobutyrate... a-aminobutyrate.... a-keto-p-methylvalerate... Isoleucine.... Methionine... Lysine... o(, e-diaminopimelate.... Pyruvate.... a-alanine... a-ketoisovalerate... Valine... a-ketoisocaproate.... Leucine.... Acetate... a-ketoglutarate.... Succinate... Fumarate... Malate... Oxalacetate... lo( 8 2( 2( 2(!! lo( lo( 71 lo( lo( lo( lo( 74 lo( ) &i lo( ) 1oc 8 5 lo( ) 1oc ( ) 45 6C 5 3: j 20 IOC 5 3: j 80 IOC 3i i 1oc 3: j 1oc E i IOC : i 1oc lo( 1 5 1oc lo( ) 3c lo( ) 1oc G * See the text for details. Glu, glutamic acid; Arg, arginine; Pro, proline; Asp, aspartic acid; Thr, threonine; Iso, isoleucine; Met, mct,hionine; Lys, lysine; Ala, alanine; Val, valinc; T,eu, leucine. protein. Metabolic links among several of these amino acids have been noted by other investigators in a number of organisms. In Neurospora crassa and Bacillus subtilis, threonine and methionine have been reported

5 I. H. ABXLSON 339 to arise from a common precursor, homoserine (14, 15). Threonine has also been related to the biosynthesis of isoleucine and valine in E. coli (16) ; however, its relationship to valine has recently been questioned (17). Lysine has been postulated to arise in B. coli from o(,e-diaminopimelic acid by decarboxylation (18, 19). However, the available evidence leaves open the question whether diaminopimelic acid is a major precursor of lysine, especially since a mutant strain of E. coli which requires lysine in addition to diaminopimelic acid has diaminopimelic decarboxylase activity, whereas some wild type E. coli strains do not (18). The results of competition experiments with members of the aspartic family and several related compounds are listed in Table I. It is seen that added aspartic acid diminishes the radioactivity of the protein aspartic acid and threonine to the same extent. This finding suggests that aspartic acid is a precursor of threonine. The relatively smaller effect of aspartic acid on isoleucine, methionine, and lysine indicates that only part of the carbon in these three amino acids is derived from aspartic acid. Since homoserine has a pronounced effect on threonine but none on aspartic acid, it is inferred that homoserine is an intermediate between aspartic acid and threonine. Homoserine is also an effective competitor in the synthesis of methionine and hence is concluded to be a precursor of the latter. Threonine in turn is seen to compete in isoleucine synthesis, and so do a-aminobutyrate, a- ketobutyrate, and especially cy-keto-p-methylvalerate. It is of interest that an E. coli mutant strain has been reported to grow on any of these four competitors (I 6). Since oc-ketobutyrate can be formed from a-aminobutyrate (20) as well as from threonine (21), it is reasonable to assume that orketobutyrate is a precursor of isoleucine. cx, P-Dihydroxy-p-methylvalerate (22) and a-keto-p-methylvalerate (23, 16) have previously been shown to be on the isoleucine pathway. The accompanying metabolic scheme, which is consistent with all the competition data, may be constructed. lysine 7 Rspllrtic rrlctllioninc acid L 7 llomoacrine -+ threoninc -3 a-l~etol~ut~~rate + a, P-dihydroxy-P-mcthylvalerute * a-kcto-p-mctli)ilvaler~t,c -+ isoleucinc Pyruvic Family--In competition experiments with pyruvate, this substance was found to have some affect 011 the glutnmic and aspnrtic families; however, a more pronounced effect was observed on alanine, valine, and leucine (Table I). Pyruvic acid hence appears to be a precursor of these three amino acids. The keto analogue of valinc, a-ket,oisovalerat,e, which has been shown to be a precursor of valine in E. cobi (24, 25), competes in

6 340 AMINO ACID IN E. COLI valine synthesis as expected. In addition, or-ketoisovalerate competes in leucine synthesis to the same extent as pyruvate and is thus indicated to be a precursor of leucine. Since it has also been reported that pantoate is formed from cy-ketoisovalerate (26), this keto acid appears to be an intermediate in at least three lines of biosynthesis. The radioactivity of leucine was also found to be influenced by or-ketoisocaproate and, to a lesser extent, acetate. In analogy with the cases of isoleucine and valine, the keto analogue of leucine is thought to give rise to the latter amino acid (see below). These results may be summarized in the accompanying scheme in which (Y,p-dihydroxyisovalerate (27) has been included. alanine 7 Fyruvic acetate acid L c (Y,&dihydroxyisovalerate -+ mketoisovalerate (- 1C) + ol-ketoisocaproate 4 1 valine leucine Xerine Family--In mammals it has been established that the carbon skeletons of glycine (28) and cysteine (29, 30) are derived from that of serine. A similar situation is indicated in E. coli by the present studies: serine as competitor almost completely suppresses the radioactivity of protein serine, glycine, and cysteine, whereas glycine suppresses the radioactivity of protein glycine, but not of serine or cysteine. Moreover, serine was found to contribute to some extent to the members of the pyruvate family. This finding is consistent with the presence in E. coli of a dehydrase which catalyzes the conversion of serine to pyruvic acid (21). Threonine was also found to have an effect on glycine, but is not believed to be a major precursor of the latter. The conversion of threonine to glycine may involve loss of a 2-carbon fragment (31). The following relationships are indicated among members of the serine family. glycine 7 Serine+pyruvic acid cysteine Competition with Compounds Related to Krebs Cycle-Since the parent substances of the four amino acid families described have a metabolic connection with the Krebs cycle, competition experiments with compounds related to this cycle have been carried out. In the course of these experiments a phenomenon was observed which has also been encountered in other competition experiments: certain compounds, which from earlier evidence had been concluded to be metabolic intermediates, gave incomplete or no com-

7 P. II. ABELSON 341 petition. These observations are in harmony with other recent findings (32) and are thought to be due to the characteristics, in a broad sense, of the enzyme systems involved or to possible accessibility barriers. For this reason incompleteness or total absence of competition by a given substance does not necessarily preclude a metabolic Ale of this substance. Thus, as is evident from the data in Table I, pyruvate, while having a considerable effect on the labeling of alanine, has a relatively small effect on the functioning of the Krebs cycle. Similarly, malate and oxalacetate have small effects, and aspartate and glutamate are more effective competitors in aspartate and glutamate synthesis than the respective keto acids. Citric, cis-aconitic, and isocitric acids have no competitive effect at all. However, in cases in which competition did occur, the results were qualitatively consistent with predictions from the Krebs cycle. Thus, added acetate, ketoglutarate, and glutamate affect the labeling of glutamate more than that of aspartate, whereas added succinate, fumarate, malate, oxalacetate, and aspartate affect the labeling of aspartate more than that of glutamate. Competition with Keto Acids-As is seen from Table I, the keto analogues of glutamic acid, aspartic acid, alanine, leucine, isoleucine, and valine are more or less effective competitors in the synthesis of the corresponding amino acids. Similarly, the radioactivity of phenylalanine was found to be suppressed by its keto analogue. On the other hand, the keto analogues of arginine, threonine, and the aldehyde analogue of glycine were found to have no competitive effect. Since the keto analogues of glutamic acid, aspartic acid, and alanine as well as those of isoleucine and valine give rise to the corresponding amino acids, it is suggested that a-ketoisocaproic acid and phenylpyruvic acid are precursors of leucine and phenylalanine, respectively. Such a precursor function of the latter two keto acids would be consistent with their respective growth-promoting activity for a leucineless (20) and a phenylalanineless (33) E. coli mutant strain and with the transaminase activity of a wild type strain (20). Most of the synthetic pathways described in this paper have been the objects of intense study at this laboratory. Many other radioactive tracers have been employed, including acetate and randomly labeled amino acids, alanine, aspartic acid, glutamic acid, glycine, isoleucine, leucine, ornithine, proline, valine, serine, and threonine. Independent evidence derived from these investigations is in agreement with the pathways described in this paper. Some of these results have been reported recently (34-37). Conclusions-The results obtained illustrate that the isotopic competition method with C14-glucose as t,he sole source of radioactivity is adapted to yield a rapid survey of the metabolism of a number of amino acids in a given species. In this manner, several previously unknown metabolic relatiofiships in E. coli have been encountered and, where a comparison was

8 342 AMINO ACID IN E. COLI possible, the present results were generally found to be in good agreement with those obtained earlier by other methods. The success of the isotopic competition technique is seen to depend on the frequent occurrence of biosynthetic reactions in which exogenously supplied metabolites are utilized rather than endogenously produced ones. SUMMARY Metabolic relationships among a number of amino acids in Escherichia coli were studied by means of the isotopic competition method with Cl -glucase as the source of radioactivity. The results were consistent with the existence of a Krebs cycle in this species and provided support for the following biosynthetic sequences: (a) glutamic acid, N-acetylglutamic acid, N-acetylglutamic r-semialdehyde, Na-acetylornithine, ornithine, citrulline, arginine; (b) glutamic acid, glutamic y-semialdehyde, Al-pyrroline-5-carboxylic acid, proline; (c) aspartic acid, lysine; (d) aspartic acid, homoserine, methionine; (e) aspartic acid, homoserine, threonine, a-ketobutyric acid, a!, P-dihydroxy-P-methylvaleric acid, a-keto-@-methylvaleric acid, isoleutine; cf> pyruvic acid, alanine; (g) pyruvic acid, cr,p-dihydroxyisovaleric acid, a-ketoisovaleric acid, ol-ketoisocaproic acid, leucine; (h) a-ketoisovaleric acid, valine; (i) serine, glycine; (j) serine, cysteine; (k) serine, pyruvic acid; (I) threonine, glycine; (m) phenylpyruvic acid, phenylalanine. The author wishes to acknowledge the substantial contributions made by Dr. Henry J. Vogel to this paper. His wide knowledge of the biosynthetic pathways established by mutant studies, a substantial effort in revising the original manuscript, and stimulating discussions of the experimental results were most valuable. BIBLIOGRAPHY 1. Roberts, It. B., and Roberts, I. Z., J. Cell. and Comp. Physiol., 36, 15 (1950). 2. Cowie, D. B., Bolton, E. T., and Sands, M. K., J. Bact., 62, 63 (1951). 3. Abelson, P. H., 13olton, E. T., and Aldous, E., J. Biol. Chem., 198, 173 (1952). 4. Vogel, H. J., Abelson, P. H., and Bolton, E. T., Biochim. et biophys. acta, in press. 5. Abelson, 1. H., Bolton, E. T., and Aldous, E., J. Biol. Chem., 198, 165 (1952). 6. Cramer, F., Papierchromatographic, Weinheim, 2nd edition, 16 (1953). 7. Cederfriend, S., and Gibbs, M., Science, 110, 705 (1949). 8. Putman, E. W., and Hassid, W. Z., J. Biol. Chem., 196, 749 (1952). 9. Vogel, H. J., Abstracts, American Chemical Societ.y, Atlantic City, 43C (1952). 10. Vogel, II..I., I roc. Nat. ilrud. SC., in press. 11. Tatum, E. L., I roc. Nat. Acad. SC., 31, 215 (1945). 12. Simmonds, S., and Fruton, J. S., J. Biol. Chem., 174, 705 (1948). 13. Vogel, II. J., and Davis, B. D., J. Am. Chem. &c., 74, 109 (1952). 14. Teas, IT. J., Horowitz, N. II., and Fling, M., J. Hiol. Che?)z., 172, 651 (1948). 15. Teas, II. J., J. Bact., 59, 93 (1950).

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10 AMINO ACID BIOSYNTHESIS IN ESCHERICHIA COLI: COMPETITION WITH C 14 ISOTOPIC -GLUCOSE Philip H. Abelson J. Biol. Chem. 1954, 206: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1