ASPARTATE METABOLISM AND ASPARAGINE SYNTHESIS IN PLANT SYSTEMS*

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1 ASPARTATE METABOLISM AND ASPARAGINE SYNTHESIS IN PLANT SYSTEMS* BY GEORGE C. WEBSTER AND J. E. VARNERt (From the Kerckhoff Laboratories of Biology, California Institute of Technology, Pasadena, California) (Received for publication, December 6, 1954) The synthesis of glutamine according to the reaction Glutamate + ATP + NH 3 e glutamine + ADP + Pi has been well established in plant (1, 2), animal (3, 4), and bacterial (5, 6) systems.l However, as several investigators have reported (3, 4, 6), when aspartate has been substituted for glutamate in the above reaction, no evidence for asparagine synthesis could be obtained. Particularly noteworthy is the report of Elliott (4) that extracts of lupine seedlings, which form considerable glutamyl hydroxamate in the presence of glutamate, NH20H, ATP, and magnesium ions, fail to form any aspartyl hydroxamate on substitution of aspartate for glutamate. In contrast to this, Black and Gray (7) have demonstrated that the formation of aspartyl hydroxamate is catalyzed by a yeast enzyme which normally forms aspartyl phosphate (but apparently not asparagine) by the following reaction Aspartate + ATP - aspartyl phosphate + ADP Despite the considerable interest in the manner of asparagine synthesis, there has been but little information on the nature of the reaction. Etiolated lupine seedlings have been reported (8) to accumulate as much as 25 per cent of their dry weight in the form of asparagine, and, as the work of both Vickery and Pucher (9) and Meiss (10) has demonstrated, provide excellent experimental material for the study of asparagine synthesis. The present paper reports studies with both intact lupine seedlings and cell-free extracts. The results indicate that asparagine can be formed in these systems by a reaction analogous to that involved in glutamine formation. * Supported in part by the Polychemicals Department, E. I. du Pont de Nemours and Company, Inc., and by a grant-in-aid to one of us (J. E. V.) from the Charles F. Kettering Foundation. Presented before the annual meeting of the American Society of Plant Physiologists, Gainesville, Florida, t Present address, Department of Agricultural Biochemistry, The Ohio State University, Columbus, Ohio. 1 The following abbreviations are used: ATP, adenosine triphosphate; ADP, adenosine diphosphate; Pi, orthophosphate; Tris, tris(hydroxymethyl)aminomethane. 91

2 92 ASPARAGINE SYNTHESIS IN PLANTS EXPERIMENTAL Lupine seedlings were grown and homogenates prepared from these and other tissues as described previously (11). Extracts of wheat germ and of wheat germ acetone powder were also prepared as described earlier (12). Ethanol precipitation of the enzyme protein was carried out by mixing 3 volumes of ethanol with the cell-free extract at l, removing the protein by centrifugation, and dissolving it in 0.5 volume of 0.1 M Tris-HCI (ph 7.5). Ethanol was removed from the protein-free supernatant fluid with a low temperature rotating evaporator. The composition of the various reaction systems is described with Tables I to III and Figs. 1 to 6. The formation of hydroxamates of aspartate and other amino acids was determined by the method of Lipmann and Tuttle (13). The formation of radioactive aspartate and radioactive asparagine was measured by their chromatographic isolation in the following manner. The reaction systems (deproteinixed with 6 per cent trichloroacetic acid and containing 5 mg. each of carrier aspartate and asparagine) were subjected to a preliminary separation on a 1 X 30 cm. Dowex 50 column. With 1.5 N HCl as the eluent, aspartate and asparagine migrate as a single peak in the position normally observed for aspartate under these conditions. This treatment serves to separate aspartate and asparagine from organic acids formed by deamination of aspartate and from,&ala&e formed by aspartate decarboxylation. The combined fractions containing aspartate and asparagine were evaporated to dryness under vacuum in a low temperature, rotating evaporator and taken up in a small volume of 0.2 M ammonium acetate of ph 5.0. Aspartate and asparagine were separated from each other on a 1 X 30 cm. column of Amberlite IR4B with 0.2 M ammonium acetate (ph 5.0) as the eluent (14). A typical separation is presented in Fig. 1. The radioactivities of the asparagine and aspartate fractions were determined by standard techniques with an end window Geiger-Miiller tube and scaling circuit. Preparation of p-alan~lamide-&bromopropionyl chloride was added slowly with constant stirring to a lo-fold excess of concentrated ammonium hydroxide. The solution was allowed to stand overnight at room temperature, concentrated t.o a thick syrup by reduced pressure distillation, and precipitated with ethanol. The product was then recrystallized three times from ethanol. Results Early experiments confirmed the reports of previous investigators (3, 4, 6) that aspartyl hydroxamate is not formed by tissue extracts under the conditions normally optimal for glutamyl hydroxamate formation. If,

3 G. C. WEBSTER AND J. E. VARNER 93 however, the concentrations of both aspartate and hydroxylamine are increased about lo-fold, it then becomes possible to demonstrate the formation of aspartyl hydroxamate. Table I illustrates that a variety of plant tissue extracts are capable of cata.lyzing the formation not only of aspartyl but also of p-alanyl hydroxamate. It should be noted that all of the components of the reaction system, amino acid, hydroxylamine, ATP, magnesium ions, and tissue extract, are necessary for optimal hydroxamate formation. The ability of the various extracts to bring about hydroxamate formation varies widely, the highest activity being found in wheat germ extracts and the lowest (no detectable activity) in spinach leaf extracts. $ SO- S Q ASPARAGINE p w 2.0- > i= 4 l.o- E ASPARTATE FRACTION NUMBER FIG. 1. Separation of asp&ate and asparagine on an Amberlite IR-4B column. 2 ml. fractions were collected from a 1 X 30 cm. column with 0.2 M ammonium acetate of ph 5.0 as the eluting agent. The fact that p-alanine is activated suggests that asparagine formation might proceed by the initial formation of p-alanylamide, followed by the carboxylation of this amide to form asparagine. The data of Table II indicate, however, that this is not the case. The carboxylative formation of asparagine from /3-alanylamide is considerably less than the formation of asparagine from free 8-alanine, indicating that the amide is probably hydrolyzed to free /3-alanine before conversion to asparagine. The direct conversion of p-alanylamide to asparagine, therefore, seems unlikely. The results of the carboxylation experiments instead suggest that aspartate itself is a precursor of asparagine. This suggestion is supported by the data of Fig. 2, in which intact lupine seedlings were incubated with ammonium chloride and aspartate-cl4 for 3 hours. The major fraction of radioactivity in the cells was found in asparagine and considerably less in other fractions (including aspartate itself).

4 94 ASPARAGINE SYNTHESIS IN PLANTS The transformation of aspartate-cl4 to asparagine-cl4 can be demonstrated with cell-free extracts of either lupine seedlings or wheat germ. As is illustrated in Table III, wheat germ extracts readily carry out asparagine synthesis in the presence of aspartate, ATP, and ammonium and magnesium ions. Almost identical results have been obtained with lupine seedling TABLE Formation of Aspartyl Hydroxamate by Extracts of Various Plant Tissues* I Tissue Aspartyl hydroxamate formedt,%alanyl hydroxamate formedt Lupine seedlings Germinating peas Wheatgermf Spinach leaves% Yea&$ * Assay system, 0.05 M Tris-HCl (ph 8.3), 0.10 M amino acid, 0.40 M hydroxylamine, 0.01 M MgS04,0.005 M ATP, and 0.5 ml. of tissue extract in a total volume of 4 ml. i Micromoles per hour per mg. of protein at 38. $ Acetone powder. TABLE Carboxylation Reactions Involving &Alanine and P-Alanylamide system* II Aspartate c.p.m. &Alanine + CY CY*Oz. 5,160 Asparagine c.p.m * The complete system contained 0.05 M Tris-KC1 (ph 7.5), 0.02 M amino acid or amide, 0.01 M NaHC140s, 0.01 M MgSO M NH&l, M ATP, 0.01 mg. each of biotin, thiamine pyrophosphate, and pyridoxal phosphate, and 0.5 ml. of wheat germ extract in a total volume of 2 ml. extracts. Even in these undialyzed preparations, requirements for ATP, NH4C1, and MgS04 are apparent. The strong inhibition of synthesis caused by cyanide and dinitrophenol suggests the participation of oxidative phosphorylation in the synthetic process, presumably in the regeneration of ATP. The strong inhibition caused by p-chloromercuribenzoate indicates a participation of sulfhydryl groups in the synthetic reaction. It appears, therefore, that asparagine can be formed by these cell-free extracts by a process similar to that involved in glutamine synthesis. The ethanol-precipitated protein forms asparagine less actively (in the

5 G. C. WEBSTER AND J. E. VARNER 95 presence of aspartate, ATP, NH&l, and MgS04) than the crude enzyme extract (Table III). When the protein-free supernatant fluid (after re- ASPARAGINE ASPARTATE TOTAL ACTIVITY INCORPORATED CTS./MIN ORGANIC ACIDS PROTEIN UNACCOUNTED FOR FIG. 2. Incorporation of the radioactivity of aspartate-crd into various fractions of 5 day-old etiolated lupine seedlings incubated for 3 hours at 38 with 0.05 M aspartate-cl4 (uniformly labeled), containing a total activity of 850,000 c.p.m., and 0.03 M NH&l. Aspartate and asparagine were isolated as described earlier. Protein was isolated by precipitation with trichloroacetic acid and treated as described elsewhere (12). The fraction designated as organic acids contains the total ethersoluble fraction of the cellular material. TABLE Characteristics of Asparagine-Forming System oj Wheat Germ Extracts III system* Asparagine-Cl4 formed Complete No NH&l * ATP MgSOa I supernatantfluid enzyme HCN dinitrophenol......_..._ p-chloromercuribenzoate ~mole * The complete system contained 0.08 M Tris-HCI (ph 7.5), 0.05 M aspartate-04, 0.04 M NH&I, M ATP, M MgS04,0.1 ml. of ethanol-precipitated enzyme from wheat germ extract, and 0.4 ml. of protein-free supernatant solution from enzyme purification, in a total volume of 1 ml. The inhibitor concentration in each case was M. Temperature, 38 ; time, 3 hours. Asparagine formation was determined by radioactive assay after separation of aspartate and asparagine chromatographically. moval of ethanol) is recombined with the protein, more than 90 per cent of the original activity is regained. It is evident, therefore, that some additional non-protein factor present in the extract is required for optimal

6 96 ASPARAGINE SYNTHESIS IN PLANTS activity. It is not clear as yet whether this factor participates directly in the synthetic reaction or whether it is merely a glycolytic intermediate that participates in the regeneration of ATP in a manner similar to that recently described by Yanari, Snoke, and Bloch (15) for glutathione synthesis. GLUTAMATE I I I MOLAR CONC. OF ATP MOLAR GONG. OF AMINO ACID FIG. 3 FIG. 4 FIG. 3. Dependence of aspartyl hydroxamate (AHA) synthesis on ATP concentration. The complete system contained 0.05 M Tris-HCl (ph 8.3), 0.10 M aspartate, 0.40 M hydroxylamine, 0.01 M MgS04, ATP in the concentrations given, and 0.5 ml. of an extract of wheat germ acetone powder in a total volume of 4 ml. Incubated at 38 for 60 minutes. FIG. 4. Dependence of aspartyl hydroxamate (HA) synthesis on aspartate concentration contrasted with the dependence of glutamyl hydroxamate formation on glutamate concentration. The complete system contained 0.05 M Bris-HCl (ph 8.3), 0.40 M hydroxylamine, 0.01 M MgSO1, 0.01 M ATP, aspartate in the concentrations given, and 0.5 ml. of an extract of wheat germ acetone powder in a total volume of 4 ml. Incubated at 38 for 60 minutes. Properties of System Responsible for Aspartyl Hydroxamate Xynthesis- In the light of the above evidence that asparagine is formed from aspartate in a manner at least somewhat analogous to the formation of glutamine from glutamate, the formation of aspartyl hydroxamate as described in Table I becomes of more interest. It seems possible that this reaction may be related to asparagine synthesis in the same way that glutamyl hydroxamate formation is related to glutamine synthesis. The properties of the aspartyl hydroxamate-forming system have, therefore, been examined as a prelude to purification of the enzyme which catalyzes asparagine synthesis. This makes it possible to establish in more detail how closely the properties of the aspartyl hydroxamate-forming system resemble those of the as-

7 G. C. WEBSTER AND J. E. VARNER 97 paragine-forming system. As is evident from Fig. 3, the enzymatic formation of aspartyl hydroxamate, like the formation of glutamyl hydroxamate, has an absolute requirement for ATP. As might be expected, the completeness of this requirement is only apparent when either a well dialyzed extract or an extract of an acetone powder is used as the enzyme source. Neither adenosine diphosphate nor adenosine monophosphate is effective GLUTAMATE M MOLAR CONC. OF NH,OH MAGNESIUM ION GONG. FIG. 5 FIG. 6 FIG. 5. Dependence of aspartyl hydroxamate (HA) synthesis on hydroxylamine concentration contrasted with the dependence of glutamyl hydroxamate formation on hydroxylamine concentration. The complete system contained 0.05 M Tris- HCl (ph 8.3), 0.10 M aspartate, 0.01 M MgS04, 0.01 M ATP, hydroxylamine in the concentrations given, and 0.5 ml. of an extract of wheat germ acetone powder in a total volume of 4 ml. Incubated at 38 for 60 minutes. FIG. 6. Dependence of aspartyl hydroxamate (AHA) synthesis on magnesium ion concentration. The complete system contained 0.05 M Tris-HCl (ph 8.3), 0.10 M aspartate, 0.01 M ATP, 0.40 M hydroxylamine, MgSOh in the concentrations given, and 0.5 ml. of an extract of wheat germ acetone powder in a total volume of 4 ml. Incubated at 38 for 60 minutes. in replacing ATP. The ph optimum for aspartyl hydroxamate synthesis by wheat germ extracts is at ph 8.3, while the optimum for glutamyl hydroxamate synthesis is found around ph 7.4. The enzyme system has a relatively low affinity for aspartate. This is apparent from Fig. 4, in which the hydroxamate-forming activity of wheat germ extract is presented as a function of substrate concentration and compared with a similar curve for glutamyl hydroxamate formation. Even more striking are the differences in relative affinities of the two systems for hydroxylamine, as illustrated in Fig. 5. The exceedingly high affinity of the glutamine enzyme for hydroxylamine is not at all apparent in the aspartyl hydroxamate reaction.

8 98 ASPARAGINE SYNTHESIS IN PLANTS Finally, aspartyl hydroxamate synthesis is highly sensitive to magnesium ion concentration (Fig. 6). Although aspartyl hydroxamate synthesis has an obvious absolute dependence on the presence of magnesium ions, it is also very sensitive to any increase in the concentration of these ions above 0.01 M. At Mg++ concentrations which are optimal for the synthesis of glutamine or glutamyl hydroxamate in plants, aspartyl hydroxamate synthesis is about 50 per cent inhibited. DISCUSSION There are striking differences in the properties of the enzymes responsible for the synthesis of glutamyl and aspartyl hydroxamates. Whether or not these differences are of importance to the manner of asparagine synthesis in the cell, and indeed whether the synthesis of aspartyl hydroxamate as studied here has any relation to asparagine synthesis itself, are questions that must await further investigation. The important facts are that asparty1 hydroxamate synthesis can be demonstrated and that there are considerable differences in the properties of the enzymes responsible for glutamyl and aspartyl hydroxamate synthesis. The differences are easily sufficient to explain the failure of previous investigators to detect the formation of aspartyl hydroxamate. An important result of the present investigation has been the demonstra,tion of a hitherto unreported pathway of asparagine synthesis; namely the direct amidation of aspartate in the presence of ATP in a manner that appears to be analogous to that involved in the synthesis of glutamine. Two other pathways of asparagine synthesis have been reported by other investigators. Mardashev and Lestrovaya (16) have asserted that liver extracts can form asparagine by a transamidation between glutamine and aspartate. In experiments with extracts of lupine and wheat germ, we have been unable to demonstrate any formation of radioactive asparagine from radioactive aspartate by such a reaction. It is somewhat doubtful, therefore, that the reaction occurs to any great extent in these plants. Meister and Fraser (17) have recently described the amination of a-ketosuccinamate to asparagine as catalyzed by liver preparations. It is not possible as yet to evaluate the importance of this reaction in the cellular synthesis of asparagine, because the biosynthesis of the amide bond of a-ketosuccinamate has not been demonstrated. It will be of definite interest to determine the relative importance of this reaction and the reaction described in the present paper in the general problem of asparagine synthesis in living cells. SUMMARY The synthesis of asparagine from aspartate and ammonia has been demonstrated in lupine seedlings and in extracts of these seedlings and of wheat

9 G. C. WEBSTER AND J. E. VARNER 99 germ. In the latter case, ATP and magnesium ions are essential for the synthesis. The extracts readily catalyze the formation of aspartyl hydroxamate from aspartate, hydroxylamine, ATP, and magnesium ions. An investigation of the properties of this reaction indicates that the substrate relations, ph optimum, and sensitivity of the system to magnesium ion concentration are sufficiently different from those of the glutamine-forming system to explain the failure of previous investigators to detect aspartyl hydroxamate formation. The inability of at least some plant tissue extracts to form asparagine by the carboxylation of p-alanylamide or by a transamidation with glutamine has also been demonstrated. BIBLIOGRAPHY 1. Elliott, W. H., J. Biol. Chem., 201, 661 (1953). 2. Webster, G. C., Plant Physiol., 28, 724 (1953). 3. Speck, J. F., J. Biol. Chem., 179, 1405 (1949). 4. Elliott, W. H., Biochem. J., 49, 106 (1951). 5. Elliott, W. H., and Gale, E. F., Nature, 161, 129 (1948). 6. Grossowicz, N., Wainfan, E., Borek, E., and Waelsch, H., J. Biol. Chem., 187, 111 (1950). 7. Black, S., and Gray, N. M., J. Am. Chem. Sot., 76,227l (1953). 8. Schulze, E., 2. physiol. Chem., 24, 18 (1898). 9. Vickery, H. B., and Pucher, G. W., J. BioZ. Chem., 160, 197 (1943). 10. Meiss, A. N., Connecticut Agr. Exp. Sta., Bull. 655 (1952). 11. Webster, G. C., Arch. Biochem. and Biophys., 47, 241 (1953). 12. Webster, G. C., and Varner, J. E., Arch. Biochem. and Biophys., in press. 13. Lipmann, F., and Tuttle, L. C., J. BioZ. Chem., 169, 21 (1945). 14. Hirs, C. H. W., Moore, S., and Stein, W. H., J. BioZ. Chem., 196,669 (1952). 15. Yanari, S., Snoke, J. E., and Bloch, K., J. BioZ. Chem., 201, 561 (1953). 16. Mardashev, S. R., and Lestrovaya, N. N., Doklady Akad. Nauk S. S. S. R., 78, 547 (1951). 17. Meister, A., and Fraser, P. E., J. BioZ. Chem., 210, 37 (1954).

10 ASPARTATE METABOLISM AND ASPARAGINE SYNTHESIS IN PLANT SYSTEMS George C. Webster and J. E. Varner J. Biol. Chem. 1955, 215: 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

THE ASSIMILATION OF AMMONIA NITROGEN BY THE TOBACCO PLANT: A PRELIMINARY STUDY WITH ISOTOPIC NITROGEN. (Received for publication, July 3, 1940)

THE ASSIMILATION OF AMMONIA NITROGEN BY THE TOBACCO PLANT: A PRELIMINARY STUDY WITH ISOTOPIC NITROGEN BY HUBERT BRADFORD VICKERY AND GEORGE W. PUCHER (Prom the Biochemical Laboratory of the Connecticut