J. Biochem. 85, 1309-1314 (1979)

Studies on Rat Liver Argininosuccinate Synthetase

Inhibition by Various Amino Acids

Shigeo TAKADA, Takeyori SAHEKI, Yoshikazu IGARASHI , and Tsunehiko KATSUNUMA

Department of Biochemistry, School of Medicine , Tokai University, Bohseidai, Isehara, Kanagawa 259-11

Received for publication, November 14 , 1979

Inhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6.3.4.5] are de

scribed.

1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturat

ing amounts of the substrates.

2. L-, L-argininosuccinate, L-, L-, and L- competitively

inhibited the enzyme activity at a low concentration of L-, with Kj values of 1.3•~

10-4 M, 2.5•~10-4 M, 6.7•~10-4 M, 6.3•~10-4 M, and 6.0•~10-4 M, respectively.

3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low

concentration of L-aspartate, with Kl values of 9.5•~10-4 M and 1.2•~10-3 M, respectively.w

4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and

noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively.

5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly

increased in the presence of a low concentration of L-histidine in the assay mixture.

6. The conformation of the enzyme was markedly changed by the addition of L-histidine as

judged from the CD spectrum. This change was partially reversed by incubation with L citrulline.

Argininosuccinate synthetase [L-citrulline; L- and crystallized the enzyme from rat liver and aspartate ligase (AMP-forming), EC 6.3.4.5] is studied the substrate kinetics (7). In the present thought to be a major regulatory enzyme of the paper, we report the mechanism of inhibition by urea cycle in mammals, because its total activity amino acids, especially by L-histidine and L- in liver is the least among the enzymes of the urea norvaline; the types of inhibition were found to be cycle (1, 2), and citrulline was accumulated in rat different. The latter compound is known to be liver in dietary transition experiments from 5 an inhibitor of the urea formation system (8). casein diet to 70% casein diet (3). Ratner and Rochovansky investigated the reaction mechanism MATERIALS AND METHODS and kinetic properties of argininosuccinate syn thetase using the enzymes partially purified from Materials-High protein (70%) diet was steer liver and hog kidney (1, 4, 5) and the enzyme obtained from CLEA Japan, Inc. (Tokyo, Japan). purified from bovine liver (6). We have purified Sephadex G-25, Sephadex G-200, and DEAE

Vol. 85, No. 5, 1979 1309 1310 S. TAKADA, T. SAHEKI, Y. IGARASHI, and T. KATSUNUMA

Sephadex A-50 were purchased from Pharmacia activity was measured by determining inorganic Fine Chemicals AB (Uppsala, Sweden). L- phosphate formation according to the method of Norvaline was obtained from Fluka AG, Chemische Fiske and SubbaRow (15), except that 0.5 N

Fabrik (Switzerland); L- from Fluka sulfuric acid was used in place of 5 N sulfuric acid.

AG, Buschs SG (Switzerland); ƒ¿-amino-n-butyric The reaction mixture (1.0 ml) contained 50 mm

acid from Calbiochem (San Diego, California); Tris-HCl, pH 7.5, 15 MM MgCl2, 5 MM L-aspartate,

phosphoenolpyruvate from Boehringer Mannheim 5 MM L-citrulline, 0.9 mm ATP, 20 units of pyro GmbH (Mannheim, Germany); NADH from phosphatase, and an appropriate amount of Oriental Yeast Co. (Tokyo, Japan); ATP and argininosuccinate synthetase. The reaction was amino acids from Kyowa Hakko Co. (Tokyo, allowed to continue for 5 min at 37•Ž and then Japan). All other chemicals were of analytical stopped by the addition of 2 ml of the color

grade from Wako Pure Chemical Industries (Tokyo, producing reagent. After 10 min at 37•Ž, the Japan). L-Argininosuccinate was synthesized absorbancy was measured at 660 nm. The reac

enzymatically from L-arginine and fumarate tion rate was linear for at least 30 min at 37•Ž.

according to the method of Ratner (9). Lactate Protein was determined by the method of

dehydrogenase [EC 1.1.1.27], adenylate kinase [EC Lowry et al. (16).

2.7.4.3], pyruvate kinase [EC 2.7.1.40], and pyro Measurement of Circular Dichroism (CD)

phosphatase [EC 3.6.1.1] were purified according Spectra-The samples were incubated at 37•Ž for to the methods of Racker (10), Kress et al. (11), 2 min with or without amino acids. Samples Tietz and Ochoa (12), and Cooperman et al. (13), were then cooled to room temperature (ca. 23•Ž) respectively. and their CD spectra were determined at room Preparation of Crystalline Argininosuccinate temperature using a JASCO J-20 automatic record

Synthetase from Rat Liver-Rat liver argininosuc ing spectropolarimeter. cinate synthetase was crystallized as reported

previously (6). The crystallized enzyme was RESULTS AND DISCUSSION stored at -20•Ž in the presence of 10 MM L

argininosuccinate, without loss of activity. The Inhibition of Rat Liver Argininosuccinate stored enzyme was thawed and passed through a Synthetase by Amino Acids-As shown in Table

Sephadex G-25 column equilibrated with 50 mm I, the inhibition patterns of argininosuccinate

Tris-HCl, pH 7.5, before use in all experiments. synthetase by various amino acids were studied The enzyme was stable for at least 8 h in ice. under various conditions. Column A shows that Enzyme Assay-In the standard assay method, L-argininosuccinate, L-histidine, and L-tryptophan the activity of argininosuccinate synthetase was inhibited the enzyme activity in the standard assay, measured spectrophotometrically according to the using saturating concentrations of the substrates. method of Rochovansky and Ratner (4), except These three amino acids were similarly inhibitory

that the reaction was carried out at 25•Ž. One when the concentration of ATP in the standard

unit was defined as the amount of enzyme produc assay (0.9 mm) was reduced to 0.15 mm (column

ing 1 ƒÊmol of AMP per min at 25•Ž. In the kinetic B). In contrast, when the concentration of L studies, two other methods were used. 1) The citrulline was reduced from 5.0 mm to 0.1 mm, enzyme activity was measured by determining AMP a number of amino acids inhibited the enzyme formation as follows: the reaction mixture (0.5 ml) activity (column C); strong inhibition was observed contained 50 mM Tris-HCl, pH 7.5, 5 mm MgCl2, particularly with L-norvaline, L-argininosuccinate, 240 units of pyrophosphatase, 5 MM L-citrulline, L-arginine, L-isoleucine, and L-valine. Column D 5 MM L-aspartate, 0.9 mm ATP, and an appropriate shows that, when the concentration of L-aspartate amount of argininosuccinate synthetase. The was reduced from 5.0 mm to 0.08 mm, some amino reaction was allowed to continue for 10 min at acids still inhibited the enzyme activity, but the 25•Ž. At the end of the reaction, 0.01 ml of the spectrum of inhibition differed from those shown

mixture was directly subjected to liquid chromatog in columns A, B, and C; little or no inhibition was

raphy (Shimadzu-Du Pont) for AMP determination observed with L-isoleucine, L-valine, L-serine, L as described previously (14). 2) The enzyme alanine, glycine, L-threonine, L-glutamine, L-,

J. Biochem. INHIBITION OF ARGININOSUCCINATE SYNTHETASE 1311

TABLE I. Inhibition of rat liver argininosuccinate synthetase by amino acids. The enzyme activity was measured by determining AMP formation as described in " MATERIALS AND METHODS ." The concentra tion of inhibitors added was 10 mm. Inhibitors were completely solubilized and the pH was adjusted to 7.5 before use. Reaction systems were as follows. A: 0.9 mm ATP, 5 MM L-citrulline, and 5 mM L-aspartate. B: 0.15 mm ATP, 5 mm L-citrulline, and 5 MM L-aspartate. C: 0.9 mM ATP, 0.1 MM L-citrulline, and 5 MM L-aspar tate. D: 0.9 mm ATP, 5 MM L-citrulline, and 0.08 mm L-aspartate.

Fig. 1. Double reciprocal plots of the velocity of the reaction against the concentrations of L-citrulline and L-aspartate. The enzyme activity was measured by determining inorganic phosphate formation as described in " MATERIALS AND METHODS," except that L-citrulline and L-aspartate concentrations were varied.

A: The L-citrulline concentration was varied from 0.025 to 0.5 mm. B: The L-aspartate concentration was varied from 0.025 to 0.5 mm. Inhibitors added and their concentrations were as follows: A 1, 2 MM L-valine; 2, 1 MM L-arginine; 3, 0.5 mM L-norvaline; 4, 1 rum L alanine; 5, without inhibitor. B 1, 2 mM L-arginine; 2, 10 MM L-; 3, 10 MM L-lysine; 4, 10 MM L norvaline; 5, 10 MM L-alanine; 6, without inhibitor.

examined by means of Lineweaver-Burk plots.

As shown in Fig. IA, all the amino acids listed in

column C of Table I except for L-histidine and

L-tryptophan competitively inhibited the enzyme

activity against L-citrulline. The K, values for

L-norvaline, L-argininosuccinate, L-arginine, L isoleucine, and L-valine were 1.3•~10-4 M, 2.5•~

10_4 M, 6.7:,/, 10_4 M, 6.3•~10-4 M, and 6.0•~10-4 M,

respectively. Rochovansky and Ratner (4) re

ported that L-argininosuccinate and L-arginine were competitive inhibitors of steer liver arginino

succinate synthetase with respect to L-citrulline * Less than 20% inhibition. and the K, values were 3.0•~10-4 M and 2.0•~10-2 M,

respectively. The K, value for L-arginine (6.7•~

L-phenylalanine, and L-asparagine. The other 10-4 M) of the enzyme is much lower than that

substances tested included L-, L-proline of the steer liver enzyme (2,0•~10-2 M). As shown in Fig. 1B, L-arginine, L-ornithine, and L-lysine ,ƒÀ-alanine, D-alanine, ƒ¿-ketoglutarate, fumarate, succinate, oxaloacetate, maleate, creatine, creatine also competitively inhibited the enzyme activity with respect to L-aspartate. The K, values for phosphate, pyruvate, N-acetylglutamate; little or no effect (less than 20%.) was observed under all L-argininosuccinate, L-arginine, L-ornithine, and

the experimental conditions studied. L-lysine were 9.5 x:10-4 M, 1.2•~10-3 M, 9.6•~10-3 M,

Mechanisms of Inhibition by Amino Acids and 1.1•~10-2M, respectively. L-Norvaline and

The mode of inhibition by some of the amino L-alanine uncompetitively inhibited the enzyme activity with respect to L-aspartate. Rognstad acids shown in columns C and D in Table I was

Vol. 85, No. 5, 1979 1312 S. TAKADA, T. SAHEKI, Y. IGARASHI, and T. KATSUNUMA

reported that L-norvaline inhibited the formation suggests that the mode of inhibition by L-histidine of urea by inhibiting ornithine transcarbamylase was of mixed type with respect to L-citrulline.

(8). We therefore measured the K, values for On the other hand, Dixon plots intersected at one L-norvaline of rat liver ornithine transcarbamylase point above the [I]-axis (Fig. 2C). These data and the enzyme; they were 1.1•~10-4 M and 1.3•~ suggest that the mode of inhibition by L-histidine 10-4 M, respectively. This result suggests that the is mixed-noncompetitive at low concentrations of

inhibition of urea formation by L-norvaline in rat L-histidine with respect to L-citrulline. At high

liver is not only at the step of ornithine trans concentrations (5 to 10 MM) of L-histidine all the carbamylase. Recently we obtained data suggest data obtained at various L-citrulline concentrations ing that inhibition of urea formation by L-nor deviated upward from the linear plots. This valine in rat liver was mainly at the step of suggests that high concentrations of L-histidine

(in preparation). exhibit positive cooperativity with respect to Effect of L-Citrulline Concentration on the L-citrulline. The Hill coefficients were 1.6 and Inhibition by L-Histidine-L-Histidine inhibited 1.0 at 5 mm and 0.1 MM L-citrulline, respectively. the enzyme activity under the standard assay Effect of L-Aspartate Concentration on the conditions. Figure 2A shows the relative activity Inhibition by L-Histidine-As shown in Fig. 3,

as a function of L-histidine concentration at four Dixon plots gave parallel lines but they curved fixed concentrations of L-citrulline (0.1, 0.25, 0.5, upward at high concentrations (5 to 10 mm) of 5.0 mm). The degree of inhibition by L-histidine L-histidine. This results suggests that the mode of increased in proportion to the decrease in L inhibition by L-histidine is uncompetitive with

citrulline concentration. L-Histidine is a chelat respect to L-aspartate, and L-histidine exhibits ing agent, but the inhibition by L-histidine was positive cooperativity. It is of interest that basic not reversed by the addition of a high concen or neutral amino acids (L-arginine, L-ornithine) tration of Mg". In Lineweaver-Burk plots (Fig. competitively inhibited the enzyme activity with 2B), all the plots were linear and they intersected respect to L-aspartate. The mode of inhibition at one point above the I/[S]-axis. This result by L-histidine with respect to ATP was noncom petitive. Effect of Preincubation with the Substrates on the Inhibition by L-Histidine-As shown in Fig. 4,

Fig. 2. Effect of L-citrulline concentration on the inhibition by L-histidine. The enzyme activity was measured by determining inorganic phosphate formation as described in " MATERIALS AND METHODS," except for changes in the L-aspartate, ATP, and L histidine concentrations as noted, A: Inhibition by L-histidine at various concentrations of L-citrulline. 1, Fig. 3. Effect of L-aspartate concentration on the 5.0 mm; 2, 0.5 mm; 3, 0.25 mm; 4, 0.1 mm. B: Plots of inhibition by L-histidine. The enzyme activity was 1/v versus 1/[cit] in the presence of L-histidine. L measured by determining inorganic phosphate for Histidine concentrations were as follows: 1, 10 mm; mation as described in " MATERIALS AND METH 2, 7.5 mm; 3, 5.0 mm; 4, 2.0 mm; 5, 1.0 mm; 6, 0 mm. ODS," except for the changes in L-aspartate and L C: Plots of 1/v versus [his] (Dixon plots) at various histidine concentrations. L-Aspartate concentrations concentrations of L-citrulline. 1, 0.1 mm; 2, 0.25 mm; for curves 1, 2, and 3 were 0.05, 0.5, and 5.0 mm, respec 3, 0.5 mm; 4, 5.0 mM. L-Histidine concentration was tively. L-Histidine concentration was varied from 0.5 varied from 0.5 to 10 mm. to 10 mm.

J. Biochem, INHIBITION OF ARGININOSUCCINATE SYNTHETASE 1313

Fig. 4. Effect of preincubation of argininosuccinate synthetase with the substrates on the inhibition by L histidine. The enzyme activity was measured by determining inorganic phosphate formation as described in " MATERIALS AND METHODS," except for the

presence of L-histidine. Argininosuccinate synthetase was preincubated with one of the substrates or L histidine at 37•Ž for 2 min. The concentration of substrate in the preincubation medium was equal to the concentration in the incubation medium. ƒ•, Pre incubated with L-citrulline; •~, preincubated with L histidine; • , preincubated with L-aspartate; ƒ¢, pre

incubated with ATP; •œ, not preincubated.

Fig. 5. Effects of L-citrulline and L-histidine on the CD the enzyme activity was slightly increased in the spectra of argininosuccinate synthetase. The CD presence of a low concentration (0.5 to 3 mm) spectra of the enzymes were measured as described in of L-histidine when the enzyme was preincubated "MATERIALS AND METHODS" in 50 mm Tris

with L-citrulline. When the enzyme was preincu HCI, pH 7.5, containing 5.0 mm MgCl., at room tempera bated with L-aspartate or ATP, the enzyme activity ture (ca. 23•Ž). A: Enzymes were incubated with or was not increased in the presence of a low con without L-citrulline or L-histidine at 37•Ž for 2 min. - centration (0.5 to 3 MM) of L-histidine. This result , Argininosuccinate synthetase alone; ---, incu

suggests that L-histidine affects the conformation bated with 5.O MM L-citrulline; ----, incubated with 1.0 MM L-histidine. B: The enzyme was incubated with of the enzyme and slightly increases the enzyme L-histidine then incubated with 10 MM L-citrulline. activity. - , The enzyme; ----, incubated with 1.0111M L Effects of L-Citrulline and L-Histidine on the histidine; ---, incubated with 1.0 MM L-histidine then CD Spectra of Argininosuccinate Synthetase-As incubated with 10 MM L-citrulline. CD data are pre shown in Fig. 5A, the CD spectrum of the enzyme sented in terms of the molar ellipticity [deg/(mol/dl)dml, incubated with L-citrulline differed slightly from as defined by the relationship [e]=(B•~M)/(C•~I•~10), that of the enzyme alone in that the former gave where [B] is the ellipticity, C is the concentration of the

a positive peak at 280 nm and a slight decrease enzyme (0.90-0.91 mg/ml), I is the light-path length of in negative ellipticity at 223 nm. In contrast, the the cell, and M is the molecular weight of the enzyme.

CD spectrum of the enzyme incubated with L

histidine was considerably different from that of bation of the enzyme with L-histidine, the negative the enzyme alone; both the negative ellipticity in ellipticity in the range of 220-250 nm was increased the range of 220-250 nm and the positive ellipticity to the level found on incubation with L-Citrulline, in the range of 250-320 nm were decreased by about but the positive ellipticity in the range of 250 40% and the positive peak at 280 nm seen for the 320 nm was not changed (Fig. 5B). There seems enzyme incubated with L-citrulline was not ob to be a relationship between the increase in activity served. When L-citrulline was added after incu

Vol. 85, No. 5, 1979 1314 S. TAKADA, T. SAHEKI, Y. IGARASHI, and T. KATSUNUMA in the presence of low concentrations of L-histidine 7. Saheki, T., Kusumi, T., Takada, S., Katsunuma, T., and the conformational changes of the enzyme & Katunuma, N. (1975) FEBS Lett. 58, 314-317 caused by L-histidine. The mechanism of inhibi 8. Rognstad, R. (1977) Biochim. Biophys. Acta 496, tion of the enzyme by histidine is enzymologically 249-254 interesting, although the inhibition is less strong 9. Ratner, S. (1957) in Methods in Enzymology (Colo wick, S.P. & Kaplan, N.O., eds.) Vol. 3, pp. 643-647, than that by L-norvaline. Academic Press, New York and London 10. Racker, E. (1952) J. Biol. Chem. 196, 347-365 The authors are grateful to Dr. Y. Watanabe for a 11. Kress, L.F., Bono, V.H., Jr., & Noda, L. (1966) critical reading of the manuscript. J. Biol. Chem. 241, 2293-2300 12. Tietz, A. & Ochoa, S. (1952) in Methods in En REFERENCES zymology (Colowick, S.P. & Kaplan, N.O., eds.) 1. Ratner, S. (1973) in Advances in Enzymology Vol. 5, pp. 365-369, Academic Press, New York (Meister, A., ed.) Vol. 39, pp. 1-90, John Willey and London and Sons, New York 13. Cooperman, B.S., Yuchiu, N., Bruckman, R.H., 2. Shepartz, B. (1973) Regulation of Burick, G.J., & Mckenna, G.P. (1973) Biochemistry Metabolism in Mammals pp. 120-135, W.B. Saunders 12, 1665-1669 Company, Philadelphia 14. Saheki, T., Kusumi, T., Takada, S., & Katsunuma, 3. Saheki, T., Katsunuma, T., & Sase, M. (1977) T. (1977) J. Biochem. 81, 687-696 J. Biochem. 82, 551-558 15. Fiske, C.H. & SubbaRow, Y. (1925) J. Biol. 4. Rochovansky, O. & Ratner, S. (1967) J. Biol. Chem. Chem. 66, 375-400 242,3839-3847 16. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., & 5. Rochovansky, O. & Ratner, S. (1961) J. Biol. Chem. Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 236,2254-2260 6. Rochovansky, 0., Kodowaki, H., & Ratner, S, (1977) J. Biol. Chem. 252, 5287-5294

J. Biochem.