408 Biochimica et Biophysica Acta, 701 (1982) 408-409 Elsevier Biomedical Press

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BBA 30007

NEGATIVE COOPERATIVITY IN HUMAN LIVER ARGININOSUCCINASE

NELSON CARVAJAL, MIREYA FERNANDEZ, JUAN PABLO RODRiGUEZ and JORGE MARTINEZ

[nstituto de Nutricibn y Tecnologia de los Alimentos, Universidad de Chile, Casilla 15138. Santiago 11 (Chile)

(Received September 9th, 1981)

Key words: Cooperativity," Argminosuccinase; (Human liver)

The kinetic properties of argininosuccinase (L-argininosuecinate arginine-lyase, EC 4.3.2.1) were investi- gated. Negative cooperativity was observed in the response of the enzyme to the substrate argininosuccinate and GTP behaved as a positive allosteric effector. These effects were observed in 60 mM potassium phosphate but not in 50 mM Tris-HCl. Structural changes in the protein molecule are suggested to explain previous observations of Michaelis-Menten kinetics for this enzyme.

Argininosuccinase (L-argininosuccinate argi- nine-lyase, EC 4.3.2.1), one of the urea cycle en-

• zymes, catalyzes the reversible'formation of arginine and fumarate from argininosuccinate. The enzyme has been studied from several sources [1-5], but the best-characterized is that found in beef liver [5-7]. Of particular interest is the regula- tion of the beef enzyme by negatively cooperative substrate binding and by the allosteric effector, GTP [7]. According to a recent report of O'Brien and Barr [8], the enzyme from human liver follows Michaelis-Menten kinetics and is not affected by GTP. Thus it would appear that the two enzymes are regulated by quite different mechanisms. How- ever, we have found conditions in which human liver argininosuccinase, like the bovine enzyme, exhibits allosteric properties. This is shown in the present paper.

Human liver argininosuccinase was purified by the procedure described by Ratner [9] for the steer liver enzyme. A preparation showing a single pro- tein band on polyacrylamide gel electrophoresis was obtained.

The enzyme activity was determined at 37°C by measuring the formation of fumarate from argininosuccinate at 240 nm in a thermostatically controlled spectrophotometer. The buffers used

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were 60 mM potassium phosphate (pH 7.5) and 50 mM Tris-HC1 (pH 7.5). Reactions were initiated by adding the enzyme to the substrate buffer solution previously warmed at 37°C. The initial velocity data were analyzed by double-reciprocal and Hill plots.

Argininosuccinate, GTP, Trizma base and fumarate were obtained from Sigma Chemical Co. All other chemicals were analytical grade.

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Fig. 1. Dependence of argininosuccinase activity on arginino- succinate concentration in the absence (O ©) and pres- ence (0 @) of 0.2 mM GTP. The buffer was 60 mM potassium phosphate (pH 7.5) and velocity is expressed as #tool fumarate produce per rain. (A) Lineweaver-Burk plot. (B) Hill plot.

409

The cleavage of argininosuceinate catalyzed by human liver arginosuccinase was studied in the absence and presence of GTP. In Fig. 1 the results obtained in 60 mM potassium phosphate buffer (pH 7.5) are presented as double-reciprocal and Hill plots. It is seen (Fig. 1A) that in the absence of GTP the line of the double-reciprocal plot shows a downward discontinuity in slope. This discontinuity is not observed in the presence of 0.2 mM GTP. Moreover, it is clear that the enzymatic activity is stimulated by the nucleotide. From the results in Fig. 1A, the g m values obtained in the absence of GTP were 5.4.10-SM and 1.5- 10-4M for the low and high concentrations of substrate, respectively. The value in the presence of 0.2 mM GTP was 4.5 • 10-5 M.

The Hill coefficients calculated from the plot in Fig. 1B were 1.0 and 0.56 in the absence of GTP and 1.0 in the presence of the nueleotide at a concentration of 0.2 raM.

The kinetic properties of argininosuccinase were also examined in 50 mM Tris-HCl (pH 7.5). Un- der these conditions, typical Michaelis-Menten kinetics was observed and GTP had no effect on the enzyme activity.

The results presented in Fig. 1 are expected for an enzyme which shows negative cooperativity and is stimulated by GTP. Therefore, the properties of argininosuccinase from human liver resembles those reported for the bovine enzyme [7]. A similar regulatory mechanism seems then to affect the enzymes from both sources. Negative cooperativity provides argininosuccinase with a mechanism to operate in the region of proportional control by adjusting the apparent Michaelis constant to the physiological concentration of the substrate [10,11]. In this way, an effective kinetic response to the substrate will be maintained over a wide range of concentrations. The role of GTP would be, as suggested by Rochovansky [7], to regulate the synthesis of arginine for protein formation.

With allosteric enzymes is frequently observed a transition to the more usual Michaelis-Menten

kinetics when some structural changes are induced on the protein molecule, Moreover, changes in conformation have been used as evidence for cooperativity in enzyme systems [12]. Possibly, changes produced during purification would ex- plain the results obtained by O'Brien and Barr [8]. In this connection some observations made by Rochovansky [7] with respect to the bovine en- zyme are of interest. Aged enzyme or enzyme stored for several days in very dilute solutions in the absence of a reducing agent undergoes a change to a form which shows no cooperativity and is not affected by the nucleotide GTP. We also observed a loss of the allosteric properties in some aged preparations of human liver argininosueeinase, but this point was not investigated in detail.

This research was supported by Grant B 1326 8112 from the Servicio de Desarrollo Cientifico, Artistico y de Cooperaci6n Internacional, Uni- versidad de Chile. The authors gratefully acknowl-

• edge the technical assistance of Mr. R. Campos.

References

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2 Bray, R.C. and Rather, S. (1971) Arch. Biochem. Biophys. 146, 531-541

3 Matagne, R.F. and Schlosser, J.P. (1977) Biochem. J. 167, 71-75

4 Rather, S. (1972) Enzymes, 3rd. edn., 7, 167-197 5 Schulze, I.T., Lusty, C.J. and Ratner, S. (1970) J. Biol.

Chem. 245, 4534-4543 6 Lusty, C.J. and Rather, S. (1972) J. Biol. Chem. 247,

7010-7022 7 Rochovansky, O. (1975) J. Biol. Chem. 250, 7225-7230 80'Brien, W.E. and Barr, R.H. (1981) Biochemistry 20,

2056-2060 9 Rather, S. (1970) Methods Enzymol. 27, 304-309

10 Cleland, W.W. (1970) in The Enzymes, (Boyer, P.D., ed.), 3rd edn., Vol. II, pp. 1-65, Academic Press, New York

I1 Koshland, D.E., Jr. (1970) in The Enzymes (Boyer, P.D., ed.), 3rd edn., Vol. I, p. 341, Academic Press, New York

12 Koshland, D.E., Nemethy, G. and Filmer, D. (1966) Bio- chemistry 5, 365-385