Kinetic Mechanism of Bovine Liver Argininosuccinate Lyase'

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 221, No. 1. February 15, pp. 143-14’7, 1983

Kinetic Mechanism of Bovine Liver Argininosuccinate Lyase’

FRANK M. RAUSHEL* AND RODERICK NYGAARD

Department of Chemistry, Texas A&M University, Cdlege Station, Texus 77843

Received August 2, 1982, and in revised form October 12. 1982

The kinetic mechanism of bovine liver argininosuccinate lyase has been determined at pH 7.5, 25°C. Fumarate and arginine are both noncompetitive inhibitors versus argininosuccinate. The dead-end inhibitor, succinate, is competitive versus fumarate and argininosuccinate, but noncompetitive versus arginine. Citrulline is competitive versus arginine and noncompetitive versus argininosuccinate and fumarate. The results are consistent with a random mechanism with the formation of two dead-end complexes: E - argininosuccinate - fumarate and E - argininosuccinate * arginine. No evidence was obtained for nonlinear reciprocal plots. The equilibrium constant was found to be 3.7 rnM.

Argininosuccinate lyase (EC 4.3.2.1) catalyzes the following reaction:

argininosuccinate arginine fumarrte The enzyme from beef liver has been ex- tensively studied by Ratner and her col- We have therefore initiated a complete leagues (l-4). They have purified the en- steady-state initial velocity and product zyme to apparent homogeneity and have inhibition study of argininosuccinate lyase shown the protein to be a tetramer of mo- and have established that the kinetic lecular weight 202,000 (2). The enzyme is mechanism is random. cold-labile and is protected from cold in- activation by the substrates or 50 mM MATERIALS AND METHODS phosphate buffer (2). Although this protein is a key enzyme in the metabolism of Argininosuccinate lyase was isolated from beef liver according to the procedure of Havir et al. (2) and nitrogen via the urea cycle, a complete Schulze et al. (7) through the DEAE-cellulose step to analysis of the kinetics of the reaction cat- a specific activity of 4.0 amol/min/mg at 25°C. All alyzed by argininosuccinate lyase has not buffers used in the purification contained 0.1 mb( appeared in the literature. Some prelimi- EDTA and 1.0 mM dithiothreitol. Argininosuccinate nary inhibition experiments have been re- was obtained from Sigma as its barium salt and was ported but no details have appeared (5,6). converted to the potassium salt and assayed as de- scribed by Havir et al. (2). All other reagents were 1 This work was supported by grants from the Re- obtained from either Sigma or Aldrich. search Corporation, the Robert A. Welch Foundation Enzyme assays. Argininosuccinate lyase activity (A-840), and the National Institutes of Health (AM was assayed in the forward or reverse direction by 30343). monitoring the appearance or disappearance of fu- ‘Author to whom all correspondence should be ad- marate at 2.40 nm with a Gilford 260 spectrophotom- dressed. eter and a 10 mV Linear recorder. All assays were

143 ooo3-9861/83/030143~5~3.00/0 Copyright 0 1983 by Academic Press. Inc. All righta of reproduction in any form reserved. 144 RAUSHEL AND NYGAARD

mM argininosuccinate. The Michaelis constant from a fit to Eq. [2] is 51 + 5 PM. Initial velocitl/ patterns. When arginine is varied at changing fixed concentrations of fumarate an intersecting double reciprocal plot is obtained. The kinetic constants from a fit of the data to Eq. [3] are shown in Table I. There is no indication in t any of these experiments for nonlinear re- 10 20 30 ciprocal plots. The ratio of maximal ve- -1 11 Argininosuctinote, mM locities for the forward and reverse reac- tions at pH 7.5 is 0.6. FIG. 1. Variation of activity with argininosuccinate concentration. Conditions: 50 mM N-2-hydroxyethyl- Product and dead-end inhibition. In the piperazine-N’-2-ethanesulfonic acid, pH 7.5, 100 mre forward reaction both fumarate and ar- KCl, 25°C. The velocities are in arbitrary units. ginine were found to be linear noncompetitive inhibitors versus argininosuccinate. Succinate was found to be a com- done at 25°C. Each 3-ml cuvette contained 50 mM N- petitive inhibitor versus fumarate and ar- 2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, gininosuccinate but a noncompetitive in- pH 7.5, 100 mM KCl, and substrates and inhibitors hibitor versus arginine. Citrulline was as indicated in the individual experiments. All re- competitive versus arginine and noncom- actions were initiated by the addition of arginino- petitive versus fumarate and argininosuc- succinate lyase (0.05-0.15 units) with the aid of an cinate. The kinetic constants from fits of adder-mixer. Data analysis. Reciprocal velocities were plotted the inhibition data to Eqs. [4] and [5] ap- graphically against reciprocals of substrate concen- pear in Table II. tration and the data were fitted to Eq. [2], assuming Equilibrium constant. The equilibrium equal variances for the velocities and using the For- constant for the reaction catalyzed by ar- tran programs of Cleland (8). Data for a sequential gininosuccinate lyase was determined at initial velocity pattern were fitted to Eq. [3], for com- pH 7.5 by making up reaction mixtures that petitive inhibition to Eq. [4], for noncompetitive in- were initially 0.470 mM in fumarate and hibition to Eq. [5] and for uncompetitive inhibition variable in arginine (2.5-5.0 mM). Enzyme to Eq. [6]. The nomenclature used in this paper is was added and the AM was monitored un- that of Cleland (9) til the reaction was complete. The final concentrations of the substrates and prod- PI ucts were calculated from the AAm using a millimolar extinction coefficient of 2.44 VAB [31 for fumarate (9). The equilibrium con- ’ = Ki.Kb + KbA + KSB + AB stant, defined as: VA [fumarate] [arginine] ’ = K(l + (I/Kis)) + A [argininosuccinate] VA [51 ’ = K(l + (I/Kis)) + A(1 + I/Kii)) TABLE I VA PI KINETIC CONSTANTS FROM INITIAL ’ = K + A(1 + I/Kii)) VELOCITY EXPERIMENTS’

RESULTS Substrate K MM) Ki 6~) Rel V Variation of arginirwsuccinhxte. The Argininosuccinste 0.051 f 0.005 - 100 Fumarate 0.06 f 0.01 0.65 f 0.1 variation of activity with argininosuccin- 170 ate concentration is shown in Fig. 1. As L-Arginine 0.28 * 0.04 2.9 + 0.6 can be seen from the data, the double re- * From fits to Eqs. [2] and [3] of the data at pH 7.5, 25-Z, ciprocal plot is linear from 30 pM to 1.0 100 mM KCI. KINETICS OF ARGININOSUCCINATE LYASE 145

TABLE II

PRODLJC~AND DEAD-END INHIBITION CQNSTANTF?~

Variable Fixed Inhibitor substrate substrate (mM) & (mW Kii (mW Inhibition

L-Arginine Argininosuccinate - 8.0 L 1.5 20 c 2 NC Fumarate Argininosuccinate - 0.47 + 0.09 0.90 k 0.06 NC Citrulline Argininosuccinate - 39 + 3 640 k 60 NC Succinate Argininosuccinate - 8325 - C

Succinate Fumarate Arginine, 0.75 34+2 - C Succinate Arginine Fumarate, 0.50 54f6 195 + 33 NC Citrulline Arginine Fumarate, 0.50 3.9 + 0.2 - C Citrulline Fumarate Arginine, 0.75 14 f 4 921 NC

’ From fits to Eqs. [4], [5], and [6] of the data at pH ‘7.5, 25°C. 100 mrd KCI. b The double reciprocal plots can be found in the Miniprint Supplement.

is 3.7 + 0.1 mM from an average of six these compounds gave the expected com- determinations. petitive inhibition patterns versus the compounds they were expected to mimic. Succinate and citrulline were also found DISCUSSION to be noncompetitive inhibitors versus ar- Argininosuccinate lyase from beef liver ginine and fumarate, respectively. This es- has previously been reported to have non- tablishes that the order of addition of fu- linear double reciprocal plots for the marate and arginine to the enzyme must cleavage of argininosuccinate to arginine be random since if there was an obligatory and fumarate (4). With our reaction con- order of addition to the enzyme, one of ditions we have obtained linear plots in all these inhibition patterns would have been cases for all substrates. The reason for this uncompetitive. For example, if arginine difference is not clear but the purified en- added before fumarate then succinate zyme from human liver has recently been would be expected to be uncompetitive ver- shown in two independent reports to ex- sus arginine. hibit normal Michaelis-Menten kinetics If the addition or release of arginine and (11, 12). The equilibrium constant for ar- fumarate from the enzyme was completely gininosuccinate lyase of 3.7 mM is in good random then both of these compounds agreement with the value of 3.22 mM as would be expected to be competitive in- determined by Kuchel et al. (6) but is not hibitors versus argininosuccinate since all consistent with the value of 11.4 mM at substrates and products would be able to 38°C as reported by Ratner (5). combine with free enzyme. However, both The inhibition experiments with the of these compounds are noncompetitive in- product and dead-end inhibitors are con- hibitors versus argininosuccinate. This sistent with a random Uni-Bi kinetic indicates that two dead-end complexes mechanism for bovine liver argininosuc- are able to form: Es argininosuccinate + cinate lyase. It has previously been shown arginine and Es argininosuccinate . fu- that compounds that resemble the sub- arate. The dead-end complex, Es arginino- strates in structure but do not chemically uccinate * citrulline, also forms as indi- react can be used to establish the order of cated by the noncompetitive inhibition of addition and release of substrates from the citrulline with argininosuccinate as the active sites of enzymes (13). In this study variable substrate, although the Kii is succinate has been used as a dead-end in- greater than 10 times the Ki,. Since suc- hibitor for fumarate and citrulline as a cinate is a competitive inhibitor versus ar- dead-end inhibitor for arginine. Both of gininosuccinate, a dead-end complex with 146 RAUSHEL AND NYGAARD argininosuccinate and succinate is appar- 4. ROCHOVANSKY, 0. (1975) J. Bid Ch.en~ 260.7225. ently unable to form. The active site of 5. RATNER, S. (1972) in The Enzymes (Boyer, P., ed.), argininosuccinate lyase is thus able to ac- Vol. VII, p. 167, Academic Press, New York. 6. KUCHEL, P. W., NICHOL, L. W., AND JEFFERY, commodate the binding of argininosuccin- P. D. (1975) B&him Biophys. Ada 397, 478. ate and one of the two products at the same 7. SCHIJLZE, I. T., LUSTY, C. J., AND RATNER, S. (1970) time. This situation is commonly seen with J. BioL Chem. 245.4534. kinases in which both ATP and the phos- 8. CLELAND, W. W. (1967) Advan EnzymoL 29.1. phorylated product are able to bind at the 9. CLELAND. W. W. (1963) Btihim Biqvhys Actu active site at the same time and thus give 67, 104. noncompetitive inhibition patterns (13,14). 10. ALBERTY, R. A., MASSEY, V., FRIEDEN, C., AND FUHLBRIGCE, A. R. (1954) J. Amer. Chem Sot REFERENCES 76, 2485. 11. O’BRIEN, W. E. AND BARR, R. H. (1981) Biochem- 1. HOBERMAN, H. D., HAVIR, E. A., ROCHOVANSICY, istry 20.2056. O., AND RATNER, S. (1964) J. Bid Chem 239, 12. PALEKAR, A. G., AND MANTAGOS, S. (1981)5. Bid 3818. Chem. 256, 9192. 2. HAVIR, E. A., TAMIR, H., RATNER, S., AND WAR- 13. CLELAND. W. W. (1970) in The Enzymes (Bayer, NER, R. C. (1965) J. Bid C&m 240, 3079. P. ed.), Vol. II, p. 1, Academic Press, New York. 3. LUSTY, C. J., AND RATNER, S. (1972) J. Bid Chem. 14. RAUSHEL, F. M., AND CLELAND, W. W. (1977) Bie 247.7010. chemistry 16.2169. KINETICS OF ARGININOSUCCINATE LYASE 147

MINIPRINT SUPPLEMENT

-0 I/fumarat., mM-’

Figure 1A: Double reciprocal plots for the initial velocity and product inhibition data that are presented in Tables I and II. Additional details are given in the text. Velocities are in arbitrary units. A: fumarate vs. arginine (a,b,c,d and e are 1.0, 0.31, 0.18, 0.13 and 0.10 nEl fumarate. respectively) 6: arginine vs. argininosuccinate (a,b,c and d are 0, 3.0, 6.0,and 9.0 HI arginine, respectively) C: fumarate vs. argininosuccinate (a,b,c and d are 0, 0.3. 0.6.and 0.9 rM4 fumarate, respectively) D: citrulline VS. argininosuccinate (a,b$ and dare 0, 50, 100 and 150 ni4 citrulline. respectively) E: succinate vs. argininosuccinate (a,b,c and d are 0. 40, @I and 120 nH succinate, respectively) F: succinate vs. fumarate (a,b c and d are 0, 3U, 60 and 90 ti succinate, respectively) G: succinate VS. arginiw (a,b,c and d are 0, 30, 60 and 90 Ml succinate, respectively) H: citrulline vs. arginine (a,b,c,d,e and f are 0, 2.0, 4.0, 6.0, 8.0 and 10.0 nM citrulline, respectively) I: citrulline vs. fumarate (a.b,c,d and e are 0, 2.5, 5.0, 7.5 and 10.0 n#4 citrullire, respectively)