The utilization of glutamine and asparagine peptides by microorganisms

The

Utilization

of G l u t a m i n e

and

by Microorganisms

Asparagine

Peptides

I

H e r b e r t K. Miller 2 and Heinrich Waelsch From the Department of Biochemistry, College of Physicians and Surgeons, Col~mbia University, New York City, New York; and from the New York State Psychiatric Institute, New York City, New York Received July 27, 1951

INTRODUCTION Although the utilization of glutamine and asparagine b y microorganisms has been studied extensively in recent years (1-8), no information as to the metabolism of their simple peptides is available. The results of c o m p a r a t i v e studies of the utilization of glycylglutamine, glutaminylglycine, glycylasparagine, asparaginylglycine, glutamine, asparagine, glutainic acid, and aspartic acid b y Lactobacillus arabinosus and Leuconosloc mesenteroides are reported in this paper. EXPERIMENTAL The syntheses and the properties of the four peptides and of the glutamine used were described in the preceding p a p e r (9). Asparagine, aspartic acid, and glutamic acid were commercial preparations of high purity. Microbiological Techniques For the study of the metabolism of the glutamine peptides, Lactobacillus arabinosus 17-5 and the unmodified glutamic acid-free medium of Hac, Snell, and Williams (6) were employed. When the dependence of growth upon the concentration of the compounds was investigated, the incubation was carried out for 20 hr. in 25 X 100 mm. test tubes loosely plugged with cotton. The total volume was 2.5 ml. which was 1This work was supported by grants from the Rockefeller Foundation and the National Vitamin Foundation. 2 Atomic Energy Commission Predoctoral Fellow, 1949-50. This report is from a dissertation submitted by Herbert K. Miller in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University. 184

UTILIZATION OF GLUTAMINE AND ASPARAGINE PEPTIDES

185

diluted to 7.5 ml. with water for turbidimetric measurement. When the dependence of growth upon time was investigated, the incubations were carried out in matched cuvettes using 6.0 ml. of single-strength medium, an a m o u n t which permitted direct reading a t appropriate time intervals. If not otherwise indicated, the temperature of incubation was 35 ~. For the study of the metabolism of the asparagine peptides, Leuconostoc rnesenteroides was grown on the asparagine and aspartic acid-free media of both Hac and Snell (7) and Camien and D u n n (8). Laclobacillus arabinosus was grown on the medium of Hac, Snell, and Williams (6). The experiments were carried out in the same manner as for the glutamine peptides. Since aspartic acid is not synthesized by L. arabinosus at 39 ~ or at 26 ~ in vacuo over K O H (10), these conditions were used for this microorganism. Since some of the amides are quite unstable to autoclaving, conditions were standardized b y adding all amidated compounds to the previously autoclaved medium aseptically. In any set of experiments the size of the inoculum was constant. Growth was measured turbidimetrically in a Coleman jr. spectrophotometer, model 6, with 19 X 105 nun. cuvettes and expressed as optical density (D). RESULTS AND DISCUSSION

It has been shown first by Hac, Snell, and Williams (6), and repeatedly confirmed, that the maximal growth response of L. arabinosus is approximately the same for equimolar amounts of glutamic acid and glutamine (1,3,11). However, when glutamic acid is used, the growth of the microorganisms shows a considerable lag period which is not observed in the case of glutamine. This finding was taken to mean that 0.4

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Fro. 1. Growth response of L. arabinosus to glutamine, glutamic acid, and glutamine peptides. X, Glutamic a c i d ; . , glutamine; A, glycylglutamine; V, glutaminylglycine. Incubated 20 hr. a t 35~ 2.5 ml. of the medium was diluted to 7.5 ml. before reading.

186

H E R B E R T K. M I L L E R AND H E I N R I C H W A E L S C H

the partial conversion of glutamic acid to glutamine represents one of the essential metabolic pathways of glutamic acid metabolism and that the lag period corresponds to the time necessary for the formation of glutamine (6). The essential nature of glutamine for the metabolism of microorganisms is further demonstrated by the finding that this amide is an obligatory metabolite for some microorganisms while in 0.5 i(.~ x x x

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ramie acid, and glutamine peptides. • Glutamic acid 1.31 #moles; O, glutamic acid 0.98 umole; e, glutamine 1.35 ~moles; -t-, glutamine 1.01 umoles; |, glutamine 0.34 ~mole; m, glycylglutamine 1.23 ~moles; V, glycylglutamine 0.31 #mole; A, glutaminylglycine 1.33 ~moles; >, glutaminylglycine 0.33 ~mole. Six ml. of medium was incubated in direct reading tubes at 35~. the case of others it has been shown by the aid of structural analogs that the presence of some of the glutamic acid in the form of glutamine is a necessary metabolic requirement (1-5,11). The equivalence of glutamic acid and glutamine with respect to maximal growth response of L. arabinosus can be seen in Fig. 1. The

U T I L I Z A T I O N OF GLUTAMINE AND ASP A R A G IN E P E P T I D E S

187

slight difference between the curves for glutamic acid and glutamine, respectively, is probably due to decomposition of some of the amide to pyrrolidone carboxylic acid which cannot be utilized. Glutaminylglycine and glycylglutamine can replace the free amide mole for mole in the metabolism of this microorganism. The similarity in behavior of glutamine and the peptides is again brought out clearly in the growth curves (Fig. 2). At higher concentrations the growth curves of glutamine and both the peptides coincide. At lower concentrations the lag period for the peptides is slightly greater than that for glutamine, but still is very much shorter than that for glutamic acid (Fig. 2). This finding is interpreted as an indication of the time needed by the microorganisms to split the peptides into glutamine and glycine. Since both glutamine peptides studied are utilized to the same extent regardless of whether the amide is substituted on the a-carboxyl or a-amino group, it appears that they serve as a source of free glutamine which is rapidly liberated by enzymatic action. It is noteworthy that the two peptides are biologically equivalent, although glutaminylglycine is chemically as unstable as glutamine, whereas the substitution on the amino group in glycylglutamine confers considerably higher stability to the amide group of this peptide. It has been shown some time ago that the sulfoxide derived from methionine (MSO) competitively inhibits the utilization of glutamic acid in the metabolism of L. arabinosus but does not interfere with the metabolism of glutamine (1). These peptides simulate the behavior of glutamine in this respect as well (Table I). When mixtures of glutamine and the peptides were used, the growth curves were indistinguishable from the growth curves obtained with TABLE I

The Effect of the Sulfoxide Derived from Methionine (MSO) on lhe Utilization of Glutamine Peptides Bacterial M

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0.22 0.22 0.22 0.22 0.23 0.23 0.22 0.22

0.16 0.01 0.13 0.13 0.14 0.14 0.14 0.15

Inhibition %

94 0 0 0

188

HERBERT K. MILLER AND HEINRICtt WAELSCH

equimolar amounts of pure glutamine (Fig. 3). On mixtures of glutamine and glutamic acid (1 : 3) the lag period is considerably shorter than that found with glutamic acid alone, though not quite as short as with glutamine alone. The same growth curve was obtained when the glutamine was replaced by equimolar amounts of either of the peptides (Fig. 4). Whereas these experments show that glutamine and its peptides are not differentiated by the microorganisms, a different picture is obtained when the utilization of asparagine and its peptides, asparaginylglycine Q5

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FIG. 3. Growth curves showing the response of L. arabinosus to mixtures of glutamine and its peptides, e, glutamine 1.35 ttmoles; 9 glutamine 1.01 ttmoles + glycylglutamine 0.31 #mole; X, glutamine 1.01 gmoles + glutaminylglycine 0.33 ttmole. Six ml. of medium was incubated in direct reading tubes at 35 ~

and glycylasparagine, is compared. Asparagine is utilized significantly less than aspartic acid by Leuc. mesenteroides (7,12) (Figs. 5 and 6). It is very striking that the two asparagine peptides simulate the behavior of the free dicarboxylic acid and not that of the amide. The growth response to small concentrations of aspartic acid is slightly better than that for equimolar amounts of the peptides, but at higher concentrations this difference appears to be eliminated. From the growth curves for Leuc. mesenteroides on low concentrations of the compounds, it may

UTILIZATION OF GLUTAMINE AND ASPARAGINE PEPTIDES

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FIG. 4. Growth curves showing the response of L. arabinosus to mixtures of gluramie acid with glutamine and its peptides, it, Glutamine 1.35 ~moles; 9 glutamic acid 1.31 ~moles; v, glutamine 0.34 ~mole -4- glutamic acid 0.98 umole; &, glycylglutamine 0.31 #~mole -F glutamic acid 0.98 ~mole; • glutaminylglycine 0.33 umole -fglutamic acid 0.98 ~mole. Six ml. of medium was incubated in direct reading tubes at 35 ~.

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190

HERBERT K. MILLER AND HEINRICH WAELSCH

be seen that the lag period is slightly shorter when the peptides are used, but that the maximal growth is about two-thirds of that obtained with equimolar amounts of aspartic acid (Fig. 7). When mixtures of aspartic acid or asparagine and the peptides were tested, the growth response appeared to be additive (Fig. 8). The presence of aspartic acid or asparagine peptides did not influence the utilization of the free amide, while the presence of the free amide did not modify thegrowth response to aspartic acid or the peptides. This may be interpreted as meaning that in the metabolism of these microorganisms there is no close relationship, as in the case of glutamic acid and glutamine, between aspartic acid or the peptides on the one hand and asparagine on the other. O.3O

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FIG. 7. Growth curves showing the response of Le~c. mesenteroides to asparagine, aspartic acid, and asparagine peptides. A, Aspartic acid 0.56 umole; • aspartic acid 0.37 i~mole; e, aspartic acid 0.19 ~mole; O, asparagine 0.56 ~mole; V, glycylasparagine 0.57 ~mole; l , glycylasparagine 0.19 ~mole; A, asparaginylglycine 0.57 ~mole; v, asparaginylglycine 0.19 ~mole. Six ml. of the medium (Camien and Dunn) was incubated in direct reading tubes at 35 ~.

It appeared highly desirable to test the striking behavior of the asparagine peptides with another microorganism. Although L. arabinosus can synthesize aspartic acid when incubated at 35 ~ at an incubation temperature of 39 ~ or at 26 ~ in vacuo over KOH it can be made to depend completely upon an outside source of the dicarboxylic acid (10). Apparently under these experimental conditions, the CO2 needed by these microorganisms for aspartic acid synthesis was lowered below the critical concentration. The growth response of L. arabinosus to aspartic acid, asparagine, and the peptides was tested under these conditions. Because, of the experimental difficulties involved, growth-time

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studies could not be carried out. Although asparagine is somewhat better utilized b y L. arabinosus than b y Leuc. mesenteroides (Figs. 5 and 6), it still does not approach in activity either aspartic acid or the peptides. Aspartic acid and the peptides are again roughly equivalent (Fig. 9). Up to the present time only a few studies on the utilization of welldefined peptides b y microorganisms are available (13-16). Agren (13) has found in a s t u d y of a variety of leucine and valine peptides t h a t the growth response b y lactic acid-producing microorganisms to these peptides was sometimes lower than to the parent amino acid but never significantly higher, These results were interpreted as indicating the

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FiG. 8. Growth curves showing the response of Leuc. mesenteroidesto mixtures of asparagine, aspartie acid, and asparagine peptides. A, Aspartie acid 0.56 ~mole; V, aspartic acid 0.37 umole -t- glycylasparagine 0.19 umole; [-1, aspartic acid 0.37 ~mole W asparaginylglycine 0.19 ~mole; • aspartic acid 0.37 ~mole; O, aspartic acid 0.37 ~mole ~ asparagiae 0.19 ~mole;' e, aspartic acid 0.19 #mole; V, aspartic acid 0.19 ~mole -~ asparagine 0.37 ~mole; T, asparagine 0.37 ~mole ~ glycylasparagine or asparaginylglyeine 0.19 ~mole; l , glycylasparagine 0.19 ~mole; v, asparaginylglycine 0.19 #mole; 9 asparagine 0.56 ~mole. Six ml. of medium (Camien and Dunn) was incubated in direct reading tubes at 35~ utilization of leucine and valine after liberation from the peptide, and t h a t low or no growth response to certain peptides m a y be referable to the absence or weakness of a specific peptidase system. Similar findings were reported by F r u t o n et al. for m u t a n t strains of Escherichia coli when phenylalanine, tyrosine, leucine, methionine, and their peptides were tested on appropriate deficient mutants (15,16). Only with proline

192

H E R B E R T K. M I L L E R AND H E I N R I C H W A E L S C H

peptides was there found a significantly higher growth response to the peptide than to the free imino acid (15). Gale has reported a similar situation with arginine peptides (14). Our findings with the glutamine peptides seem to be best understood by assuming t h a t glutamine is liberated from these peptides and used as such just as in the cases mentioned above in which peptides were used to the same extent as the unsubstituted amino acids. The preferred utilization of asparagine peptides over asparagine cannot be interpreted with any degree of certainty because of our lack of knowledge of the enzyme systems involved in the metabolism of aspartic acid, asparagine, and their peptides. At present there is no metabolic .

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FIG. 9. The growth response of L. arabinosus at 39~ and at 26~i n vacuo over KOH to asparagine, aspartic acid~ and asparagine peptides, o, Aspartic acid; A, asparagine; O, glycylasparagine; X, asparaginylglycine; solid line, 22 hr. incubation at 39~ broken line, 22 hr. incubation at 26~ i n vacuo over KOH. Two and one-half ml. of the medium was diluted to 7.5 ml. before reading. reaction known by which the amide could be utilized before being degraded to aspartic acid with the exception of those enzymatic exchange reactions which effect the exchange of the amide group with other amines (17). Gale (14) and Fruton (15,16) suggested the possibility, as an explanation for the better utilization of the arginine and proline peptides, respectively, that they are not attacked b y the enzymes which degrade the free amino acids and that they are thus conserved for anabolic processes. Such an explanation is unlikely in the case of the asparagine peptides since these and also aspartic acid are utilized more readily than is asparagine. On the basis of the experi-

UTILIZATION OF GLUTAMINE AND ASPARAGINE PEPTIDES

193

mental evidence presented it also appears unlikely that the poor utilization of the amide is caused by its interference with the utilization of aspartic acid derived from it (Fig. 8). An explanation for the comparable utilization of the asparagine peptides and the poor utilization of the free amide may be seen in the possibility that asparagine is deamidated at a faster rate when bound in peptide linkage than when free. Preliminary experiments indicate that such a situation holds true for acid hydrolysis. Aspartic acid may then be liberated from the deamidated peptides by enzymatic action. The general significance of the proposed mechanism would lie in the fact that it suggests that certain amino acids (e.g., asparagine) may, under special conditions, be metabolized with greater ease or by a different pathway when bound in peptide linkage. Such a mechanism may also explain why arginine (14) and proline peptides (15,16) are better utilized than the respective free amino acids. The asparagine peptides appear to be particularly useful tools for the study of peptide metabolism since we have available two reference points of metabolic response, that to the free amide and that to aspartic acid. A high rate of penetration of the peptides and of aspartic acid as opposed to a low rate of penetration of asparagine into the microbial cell might also account for the differential response. However, available evidence, although limited only to glutamine and glutamic acid up to now, indicates that this amide penetrates into the microbial cell (18) or into mammalian tissues (19) at a faster rate than the dicarboxylic acid. It is conceivable that the two peptides fulfil a need for naturally occurring asparagine peptides or meet the conditions'under which the peptides themselves or the asparaginyl radical may be incorporated into proteins. SUMMARY

The utilization of glutaminylglycine and glycylglutamine by Lactobacillus arabinosus was compared with that of glutamine and glutamic acid. The growth response to these peptides in its dependence upon concentration or upon time as well as the action of these peptides toward glutamic acid antimetabolites was indistinguishable from that of glutamine. It is therefore suggested that the peptides serve as a source of free glutamine liberated by enzymatic action.

194

HERBERT K. MILLER AND HEINRICH WAELSCH

In contrast to the behavior of the glutamine peptides, asparaginylglycine and glycylasparagine are significantly better utilized than asparagine by both Leuconostoc mesenteroides and L. arabinosus. Their utilization approximates that of aspartic acid. The significance of this finding is discussed. REFERENCES 1. WAELSCH,H., OWADES,P., MILLER, ~[. K., AND BORER, E., J. Biol. Chem. 166, 273 (1946). 2. BOREK, E., AND WAELSCH,H., g. Biol. Chem. 177, 135 (1949). 3. MCILWAIN,I{., FILDES,P., GLADSTONE,G. P., ANDKNIGHT,B. C. J. G., Biochem. J. 33, 223 (1939). 4. ROPER, J. A., AND MCILWAIN,H., Biochem. J. 42, 485 (1948). 5. MCILWAIN,H., ROPER, J. A., AND HUGHES, D. E., Biochem. J. 42, 492 (1948). 6. I~AC,L. R., SNELL, E. E., AND WILLIAMS,m. J., J. Biol. Chem. 159, 273 (1945). 7. HAc, L. R., AND SNELL, E. E., J. Biol. Chem. 159, 291 (1945). 8. CAMmN, M. N., AND DUNN, M. S., Proc. Soc. Exptl. Biol. Med. 75, 74 (1950). 9. MILLER, H. K., AND WAELSCH,H., Arch. Biochem. Biophys. 35, 176 (1952). 10. BORER, E., AND WAELSCH,H., J. Biol. Chem. 190, 191 (1951). 11. POLLACK,M. A., AND LINDNER, M., J. Biol. Chem. 143, 655 (1942). 12. DUNN, M. S., SHANKMAN,S., CAMIEN, M. N., FRANKL, W., AND ROCKLAND, L. B., J. Biol. Chem. 156, 703 (1944). 13..~GRE~, G., Acta Physiol. Scand. 13, 347 (1947). 14. GALE, E. F., Brit. J. Exptl. Path. 26, 225 (1945). 15. FRUTON, J. S., AND SIMMONDS,S., Cold Spring Harbor Symposia Quant. Biol. 14, 55 (1949). 16. TAYLOR,S. P., SIMMONDS,S., AND FRUTON, J. S., J. Biol. Chem. 187, 613 (1950). 17. GROSSOWiCZ,N., WAINFAN,E., BORER, E., AND WAELSCH, H., J. Biol. Chem. 187, 111 (1950). 18. GALE, E. F., J. Gen. Microbiol. 1, 53 (1947). 19. SCHWERIN,P., BESSMAN,S. P., ANDWAELSCH,H., J. Biol. Chem. 184, 37 (1950).