Eur. J . Biochem. 75, 571-581 (1977)

L-Ornithine Carbamoyltransferase from Saccharomyces cerevisiae : Steady-State Kinetic Analysis Jean-Paul SIMON and Victor STALON

Laboratoire de Microbiologie, Faculte des Sciences, Universite Libre de Bruxelles, and Institut de Recherches du Centre d’Enseignement et d e Recherches des Industries Alimentaires et Chimiques, Bruxelles

(Received December 8, 1976)

Ornithine carbamoyltransferase of Saccharomyces cerevisiae is subjected to an enzymatic regulation of its anabolic activity when it is bound to the inducible catabolic arginase as described earlier. This regulatory ornithine carbamoyltransferase essentially catalyzes the synthesis of citrulline, but the reverse reaction could be demonstrated using arsenate instead of phosphate. Steady-state initial velocity studies of the reverse reaction indicate that the mechanism is consistent with a rapid-equilibrium random model (in which all steps are in equilibrium, except that concerned with the interconversion of the central ternary complexes) involving the formation of enzyme . ornithine . arsenate and enzyme . citrulline . phosphate dead-end complexes.

In the forward direction, although the mechanism also appears to be random, the results are in better agreement with a preferred ordered binding of substrates, with carbamoylphosphate adding first. This degenerate form of the random mechanism is discussed.

The enzyme ornithine carbamoyltransferase cata- lyzes the condensation of carbamoylphosphate with ornithine to produce citrulline and phosphate. In Saccharomyces cerevisiae, this carbamoyltransferase is engaged in the biosynthetic pathway of arginine and performs a regulatory function [l - 31. Arginase, the first enzyme of the arginine catabolic pathway, has the remarkable ability to bind to ornithine carba- moyltransferase and to inhibit its activity. Because of the importance of the S. cerevisiae ornithine carba- moyltransferase as the target of this epiarginasic regulation, it is of interest to understand its kinetic mechanism of catalysis.

Several models were earlier proposed for the transcarbamylases of Streptococcus faecalis [4], E. coli [ 5 ] and the anabolic carbamoyltransferase of Pseudomonas [6], but as yet no detailed description has been given for the regulatory transferase of Saccharomyces cerevisiae. The work reported here includes the determination, in both directions, of the initial velocity pattern, product inhibition, dead-end inhibition and substrate inhibition. In the course of these kinetic studies, we have found that information from the reverse reaction, the cleavage of citrulline, tells more about the mechanism than the data obtained from the forward reaction.

Enzyme. I.-Omithine carbamoyltransferase (EC 2.1.3.3)

MATERIALS AND METHODS

Materials

Carbamoylphosphate, ornithine and norvaline were purchased from Sigma and used without further purification. Citrulline, commercial product from Sigma, was purified twice following the method of Rivard and Carter [7] to remove ornithine. [‘“CI- Carbamoylphosphate (dilithium salt) was obtained from New England Nuclear with a specific activity of 50 mCi/mol. Phosphonoacetate was kindly supplied by M. Penninckx. All other chemicals and reagents were commercial preparations of analytical grade.

All salt solutions were brought to pH 8. Ornithine carbamoyltransferase was purified extensively to the penultimate step of purification as previously de- scribed [S].

Ornithine Carbamoyltransjerase Assay

In the forward direction, ornithine carbamoyl- transferase activity was determined in one of two ways. A colorimetric measurement of citrulline pro- duction as previously described [ 5 ] was used. This assay is reproducible and was used in the range of 0.1 - 1.5 pM of citrulline. The assay mixture contained 50 mM Tris/maleate/HCl buffer, pH 8, substrate or effectors to the desired concentration and enzyme to

572 Steady-State Kinetic Analysis of L-Ornithine Carbamoyltransferase

yield a final volume of 2.0 ml. The reaction was started by addition of carbamoylphosphate after preincubation of the reaction mixture 3 min at 30 "C. The incubation time for assay was usually 10 min. The reaction was terminated by the addition of 2 ml 1 M HCI and the total reaction mixture was taken for the colorimetric citrulline analysis.

When more rigorous kinetic analysis was per- formed at a citrulline concentration below 0.1 M (not detectable by colorimetric assay), or when citrul- line was used as effector, ['4C]carbamoylphosphate assay was used [ 5 ] . The counting efficiency with a Beckman model LS 100 L scintillation counter was usually 80 %. Blanks without enzyme are essential for this assay.

Ornithine Carbamoyltransferase Assay of the Reverse Reaction

The arsenolytic cleavage of citrulline previously described [ S ] was used with slight modifications. The reaction mixture of 2.0 ml is made of 50 mM Tris/ maleate buffer pH 8 and substrates and effectors are added to the desired concentration. The reaction was started by addition of enzyme chosen so that no more than 10 % of the rate-limiting substrate was consumed.

RESULTS KINETIC STUDIES OF THE REVERSE REACTION

The formulation of Cleland [9] is used throughout this work.

Initial Velocity Patterns

When citrulline was the varied substrate at various fixed levels of arsenate, the initial velocity pattern shown in Fig.1 was obtained. The lines appear to converge at a point on the abscissa within the experi- mental error of the fitted lines. Thus the K,,, of citrul- line, found to be equal to 9mM, was independent of the concentration of arsenate. A similarly converg- ing initial velocity pattern was observed also when citrulline concentration was kept fixed at various levels and the concentration of arsenate was varied. The Michaelis constant determined was equal to 3.4 mM. Such initial velocity pattern rules out any mechanism which requires the dissociation of one product before addition of the second substrate and is thus a sequential mechanism which follows the rate equation of the general form when one substrate does not affect the binding of the other substrate.

where u is the initial velocity, P and Q are citrulline and arsenate concentrations and K terms are their Michae-

A [Arsenate] [mM)

0.5 [Arsenate].' (rnM-')

Fig. 1. Initial velocity studies in the absence of product with citrulline as variable substrate. (A) Arsenate concentrations are shown. u is the relative velocity. (B) Replot of slope (0) and [ S ] / u axis intercept (0) with respect to arsenate concentrations

lis constants. The same form of Eqn (1) is obtained whether there is an obligatory ordered addition of substrates and release of product or not. Consequently converging initial velocity patterns are given by the ordered bi-bi mechanism, the Theorell-Chance mecha- nism as well as by the random mechanism.

Although the point of intersection of Fig. 1 has no diagnostic value for distinguishing the overall kinetic mechanism, this pattern is often characteristic of a random model. Nevertheless, studies of product inhibition and of dead-end inhibition can often be used to make a choice among the various possibilities.

Dead-end Inhibition bj> Phosphonoacetate

Inhibition by phosphonoacetate was competitive versus arsenate and versus citrulline (Fig. 2). Had addition of substrate to ornithine carbamoyltrans- ferase been ordered, inhibition would have been noncompetitive or uncompetitive towards one of the substrates. Therefore, it would appear that the arseno- lytic cleavage of citrulline by the transferase of S . cerevisiae followed a random sequence of addition of

J.-P. Simon and V. Stalon 513

[ Phosphonoacetate] (mM) [Aisenate]- ' (rnM-')

[Phosphonoacetate] (mM)

C

Fig. 2. Dead-eiid inhibition by phosphonoacetate. (A) Reciprocal reverse velocity with respect to reciprocal arsenate concentration; citrulline concentrations are taken equal to 10 mM. The concen- trations of phosphonoacetate are shown. (B) Replot of slopes with respect to phosphonoacetate. (C) Reciprocal reverse velocity with respect to reciprocal of citrulline concentration. Arsenate concen- trations were taken equal to 10 mM. The concentrations of phos- phonoacetate are shown. (D) Replot of slope with respect to phosphonoacetate concentration

substrates. The rate equation for the rapid-equilibrium mechanism in reciprocal form in the presence of phosphonoacetate (I), with arsenate (Q) and citrulline ( P ) as the variable substrates is given by Eqns (2) and ( 3 ) respectively.

Eqns (2) and ( 3 ) predict that secondary plots of the slope of the initial velocity against the concentration of phosphonoacetate ( I ) should be linear. This was found to be true in the present studies. From the horizontal intercepts of these plots, the apparent inhibition constant (Kiapp) can be calculated. Eqns(2) and ( 3 ) predict also that the apparent inhibition constant thus obtained should be a function of the concentration of the non-varied substrate. This has been found to be so in the present studies (Table 1). The real Ki values have been calculated from the relations :

Inhibition by Phosphate

In these studies, phosphate, the true substrate of the reaction, was used as dead-end inhibitor. As the equilibrium constant of the reaction catalyzed by ornithine carbamoyltransferase favors citrulline syn- thesis, the phosphorolytic cleavage of citrulline re-

Table 1. Calculated values for Michaelis and inhibition constants The reaction was assumed to be random with rapid equilibrium at all steps except the interconversion of the central complex. Calculations were made by substituting into the relationships given in the text the value of the Michaelis constant for arsenate (K,J, citrulline (K,) and the fixed substrate concentration given in the text. Kis refers to the slope replot, Kil to l / v axis intercept replot and the Ki terms are the dissociation constants of the inhibitors with the free enzyme

Inhibit or ~ ~~~ ~

Variable substrate Fixed substrate Measured values Calculated values

mM

- citrulline - arsenate

Phosphonoacetate citrulline arsenate

Phosphate citrulline arsenate

Ornithine citrulline arsenate

arsenate citrulline

arsenate 10 mM citrulline 10 mM

arsenate 10 mM citrulline 10 mM

arsenate 10 mM citrulline 10 mM

K p = 9.0 Kq = 3.4

Ki, = 7.6 Ki, = 5.0

Kis = 1.7 Kis = Kii = 5.7

Ki, = 0.22 Kis = K ~ I = 0.65

Kp = 9.0 Kq = 3.4

K, = 2.06 Ki = 2

K, = 1.7 Kj = 1.4

K,b = 0.22 K,b = 0.30

574

>

Steddy-State Kinetic Analysis of 1.-Ornithine Carbamoyltransferdse

A

3

1

0

[Phosphate] (rnM) [Arsenate].’ (rnM-’)

zim

1

[Phosphate] (rnM) 0.1

[C~ t ru l l i ne ]~ ’ ( rnM- ’ )

Fig. 3. Dead-end inhibition hy phospltate. (A) Reciprocal reverse velocity with respect to the reciprocal of arsenate concentration; citrulline concentrations are taken equal to 10 mM. u is the relative velocity. Phosphonoacetate concentrations are shown. (B) Replot of slope with respect to phosphate concentration. (C) Reciprocal reverse velocity versus reciprocal of citrulline concentration. Ar- senate concentrations were taken equal to 10 mM. Phosphate concentrations are shown. u is the relative velocity. (D) Replot of slope (0) and l / u axis intercept (0) with respect to phosphate concentration

presents less than 1% of the contribution for the velocity in the studies of the arsenolysis of citrulline.

Phosphate gives linear competitive inhibition with arsenate as variable substrate; with citrulline as variable substrate, phosphate gives linear pure non- competitive inhibition (Fig. 3) . These results suggest that phosphate can react with both the free enzyme and the enzyme . citrulline complex, and the initial rate equation becomes

(4)

( 5 )

~ cn

1 [Ornithine] (rnM)

2

1

0:

0. 0

I 0.1

[Arsenate]-’(rnM-’)

1

[Ornithine] (mM) [ C i t r u l l ~ i i e ] ~ ~ ( m M ~ ~ )

Fig. 4. Product inhibi~io~i hq’ orriithine. (A) Reciprocal reverse velocity versus reciprocal of citrulline concentration; arsenale concentrations were taken equal to 10 mM. Ornithine concentra- tions are shown. (B) Replot of slope (0) and l / c axis intercept (0) with respect to ornithine concentrations. (C) Reciprocal reverse velocity versus reciprocal of arsenate concentration; citrulline concentrations were taken equal to 10 mM. Ornithine concentra- tions are shown. (D) Replot of slope with respect to ornithine concentration

where I represents the inhibitory concentration of phosphate in Eqns (4) and ( 5 ) ; Ki is the dissociation constant for the reaction of phosphate with free enzyme or with the binary enzyme-citrulline complex. Eqn (4) corresponds to a linear competitive inhibition and Eqn ( 5 ) to a linear noncompetitive inhibition. If arsenate ((2) is the varied substrate, then Eqn (4) predicts that the apparent inhibition constant Ki, is equal to its actual dissociation constant, and Eqn(5) predicts that the apparent slope or intercept inhibi- tion constant should depend on the concentration of arsenate (Q)

Ki = Kiapp ( ----). Kq Kq + Q

Ornithine Inhibition

Inhibition by ornithine was competitive with respect to citrulline and noncompetitive with respect

J.-P. Simon and V. Stalon 51 5

to arsenate (Fig. 4). Thus the inhibition by ornithine could be described by the same equation as Eqns (4) and ( 5 ) with the following substitutions of ( Q ) arsenate with ( P ) citrulline. The values of inhibition constant derived from these equations are listed in Table 1.

Kinetic Model for the Arsenolytic Cleavage of Citrulline

The results are consistent with a random mecha- nism for the kinetics of the arsenolytic cleavage of citrulline. If the mechanism of ornithine carbamoyl- transferase was ordered, phosphonoacetate should be a noncompetitive or an uncompetitive inhibitor of one of the substrates. In addition, the results suggest that the enzyme possesses distinct subsites for ar- senate and citrulline so that the dead-end complexes enzyme . arsenate . ornithine and enzyme . phos- phate . citrulline can form.

The fact that the real physiological ternary com- plex could be formed also eliminates a Theorell- Chance mechanism as proposed for E. coli ornithine carbamoyltransferase [5] .

KINETIC STUDIES O F THE FORWARD REACTION

There is no theoretical requirement for a reaction with a rapid-equilibrium random mechanism in one direction to follow a random mechanism in the opposite direction. The possibility that the reaction in the forward and pseudo-reverse directions might proceed by different mechanisms cannot be ruled out, as pointed out by Cleland in the case of creatine kinase [lo].

Initial Velocity in the Absence of Products

Fig. 5 shows that double-reciprocal plots of initial velocity versus ornithine at different constant concen- trations of carbamoylphosphate gives straight lines converging below the x axis. Secondary plots of slope and intercept against reciprocal non-varied substrate concentration were linear. Ornithine inhibi- tion was evident only at the highest substrate concen- tration and was never observed in the concentration range used in this study. The experiments at low substrate concentration established clearly that the lines are intersecting while those at higher concentra- tions (not shown) allow a better determination of the Michaelis constant. Intersecting patterns were also obtained in reciprocal plots when carbamoylphos- phate was the variable substrate. Values for the kinetic constants obtained from these plots are listed in Table 2.

Other Inhibition Studies

The initial velocity does not differentiate ordered or random mechanism, but dead-end inhibition and

:. I 0.01

A [Ornithine] (KM)

Ornithinej- ' (pM-

0.1

Fig.5. Initial velocity in the absence of product. (A) Eflect of ornithine concentration on the initial velocity of the forward reaction using the radioactive assay. Carbamoylphosphate con- centrations are shown. 11 is a relative reaction rate. (B) Slope replot. (C) l j u axis intercept replot

Table 2. Kinetic constants of ornithine carbamoyltransferase reaction as determined from initial velocity studies The values for kinetic parameters were obtained by fitting initial velocity data to equations describing the sequential mechanism according to Cleland 191. The data of Fig. 5 were used to determine K,, and Kb. K, and Kb represent Michaelis constants for carba- moylphosphate and ornithine respectively. K,, represents the dis- sociation constant for the reaction of carbamoylphosphate with free enzyme while K,b is the dissociation constant for the reaction of ornithine with free enzyme in a random mechanism

Kinetic constant Value

product inhibition can be used for further under- standing of substrate addition.

Phosphonoacetate gives a linear competitive in- hibition with respect to carbamoylphosphate (Fig. 6) and linear noncompetitive inhibition (Fig. 7) with

576 Steady-State Kinetic Analysis of L-Ornithine Carbamoyltransferase

A

10

1 >

[ P hosphonoacetate] (rnM)

0.1 [Carbarnoylphosphatej-I (FM-')

1 Phosohonoacetatej (rnMi

Fig.6. Dead-end inhibition of the forward reaction by phosphono- acetate, (A) Reciprocal plots of velocity against the reciprocal concentrations of carbamoylphosphate. Ornithine concentrations were taken equal to 0.2 mM. Phosphonoacetate concentrations are shown. (B) Replot of slope versus phosphonoacetate

respect to ornithine as variable substrate. These inhibition patterns are predicted for either a random addition mechanism or an ordered mechanism with carbamoylphosphate adding first, but rule out a compulsory ordered mechanism in which ornithine is the first substrate.

Norvaline, the best analogue of ornithine, gives linear competitive inhibition with respect to ornithine but the inhibition with respect to carbamoylphosphate is uncompetitive (Fig. 8). The observed patterns are thus consistent with a compulsory mechanism in which carbamoylphosphate adds first. The diagnosis assumes that norvaline and ornithine bind to the same enzyme form. If, in fact, the system is random with respect to carbamoylphosphate and ornithine and norvaline binds only to the binary enzyme . car- bamoylphosphate complex, then norvaline will still be competitive towards ornithine and uncompetitive towards carbamoylphosphate. In other words, the inhibition patterns of norvaline do not constitute an absolute diagnostic for an order of substrate ad- dition. Most of the ornithine analogs which have

10 Phosphonoacetate] A

IC

5

0

p ::--::::.-::.::: 10

I Orni t hinel-' irnM-'

1 Phosphonoacetate] (rnM)

Fig. 7. Dead-end inhibition of the forward reaction by phospkono- acetate. (A) Reciprocal plots of velocity against the reciprocal concentration of ornithine. Carbamoylphosphate concentrations were taken equal to 6.5 pM. Phosphonoacetate concentrations are shown

been tested for E. coli ornithine carbamoyltransferase [5] appear to be uncompetitive with respect to car- bamoylphosphate.

2,4-Diaminobutyrate, competitive inhibitor towards ornithine, clearly acts as a noncompetitive inhibitor with respect to carbamoylphosphate in our case (Fig. 9). This last result suggests that the kinetic mechanism in the forward direction is also random, as found for the arsenolytic cleavage of citrulline.

Phosphate as Product Inhibitor. The inhibition by phosphate was studied at non-saturating and at saturating concentrations of ornithine. Phosphate was found to be a linear competitive inhibitor of carbamoylphosphate at both concentrations. The Ki calculated from the replots of the slope against the concentration of phosphate were similar for both concentrations of ornithine (Table 3) . At the non- saturating concentration of carbamoylphosphate a clear-cut noncompetitive inhibition occurred (Fig. 10). This pattern could be easily explained in a random mechanism by the formation of a dead-end enzyme

phosphate . ornithine ternary complex.

J.-P. Simon and V. Stalon

F

1 . -

1 [Norvaiine] (rnM)

[Norvaline] (rnM) , I ,

1 I 5

1 . -

(Norvaline] (rnM)

/ l o

0.3 iNorvaline] (rnM; [Carbamoylphosphate]-'( FM-')

8. Dead-end inhibition of the ,forward reaction by norvaline. (A) Reciprocal plot of velocity against reciprocal of ornithine concentration; carbamolyphosphate concentrations were taken equal to 6.5 pM. Norvaline concentrations are shown. (B) Replot of slope versus norvaline concentration. (C) Reciprocal plot of velocity against reciprocal of carbamoylphosphate concentration; ornithine concentrations were taken equal to 0.2 mM. (D) Replot of 1 i c axis versus norvaline

[ 2 , 4 -Diaminobutyrate] (rnM) [ Carbarnoylphosphate]-' (FM-')

(2,4-Diaminobutyratej (mM)

Fig. 9. Dead-end inhibition by 2,4-diaminobutyrate. (A) Replot of the reverse velocity with respect to the reciprocal carbamoylphos- phate concentration. Ornithine concentrations were taken equal to 210 pM. 2,4-Diaminobutyrate concentrations are shown. (B) Replot of slope versus 2,4-diaminobutyrate concentrations. (C) Replot of the intercept at l / v axis with respect to 2,4-diamino- butyrate concentration

Table 3. Apparent kinetic constants associated with product and dead-end inhibition for the forward reaction The data were analyzed from slope or intercept with respect to inhibitor concentration

Inhibitor Varied substrate Fixed substrate Apparent Ki from

slope intercept

mM

Phosphate (Q) carbamoylphosphate (A) 0.2 2.75 k 0.2 2.5 2.2 * 0.2

ornithine (B) 0.00654 10 * 1 3.6 * 0.4 carbamoylphosphate (A) 0.2 30 2

32 k 2 ornithine 0.00654 100 * 5 42 f 4

ornithine (B) 0.00654 0.28 f 0.1 2.5 0.25 k 0.1

2.0 * 0.2 2.0 1.9 f 0.2

Citrulline (P) 40.0

Norvaline carbamoylphosphate (A) 0.2 0.41 f 0.15

Phosphonoacetate carbamoylphosphate (A) 0.2

2,4-Diaminobutyrate carbamoylphosphate 0.21 2.6 k 0.3

20 * 1 3.1 & 0.2 ornithine (B) 0.00654 70 f 5

ornithine (B) 0.00654 9.6 0.5

578 Steady-State Kinetic Analysis of L-Ornithine Carbamoyltransferase

L

[Phosphate] (rnM)

[Phosphate] (mM)

Fig. 10. Product inhibition of' the forward reaction by phosphate. (A) Reciprocal reverse velocity versus reciprocal ornithine concen- tration. Carbamoylphosphate concentrations were taken equal to 6.5 pM. Phosphate concentrations are shown. D is the relative velocity. (B) Replot of slope against phosphate concentration. (C) Replot of l / v axis against phosphate concentration

Phosphate Induces Ornithine Inhibition

The most convincing evidence that this ternary complex is formed and that the mechanism is random in the forward direction is shown by the effect of phosphate which induces ornithine inhibition (Fig. 11). This inhibition is competitive with respect to carba- moylphosphate. In an ordered mechanism, the ob- servation of appreciable inhibition by the second substrate (B) when the product (Q) is present, is explained by the fact the these metabolites mimic the substrate which they replace well enough to permit the simulation of the central ternary complex. This inhibition is always noncompetitive.

In contrast, in a random mechanism, this inhibi- tion is easily understood and is caused by the forma- tion of the enzyme. phosphate. ornithine (EBQ) abor- tive complex at high ornithine (B) levels. This resulted in 'an alternative reaction pathway since EBQ can break down to form EB which can in turn react with A to generate the central ternary complex. Such substrate inhibition is thus always hyperbolic com-

[Ornithine] (mM)

[Phosphate] = IOmM

10 [Carbamoylphosphatel-I (rnM-')

10 (Ornithine] (mM)

Fig. 11, Phospharc~-induced ornithine inhibition. (A) Reciprocal reverse velocity with respect to reciprocal carbamoylphosphate concentration. Phosphate concentrations were taken equal to 10 mM. Ornithine concentrations are shown. (B) Replot of slopes with respect to ornithine concentration

petitive versus A, since A and Q compete for the EB form. However, in this experiment (Fig. 11) ornithine seems to be a linear competitive inhibitor towards carbamoylphosphate, this inhibition is clearly limited by growing concentrations of ornithine. On the other hand, if the majority of the reactive flux goes through the pathway in which carbamoylphos- phate is the first-bound substrate, ornithine inhibition induced by phosphate should appear almost linear.

Citrulline as Product Inhibitor

At nonsaturating ornithine (B) concentrations, citrulline (P) is a competitive inhibitor towards car- bamoylphosphate (A) (Fig. 12), and this inhibion is completely overcome by a saturating concentration of carbamoylphosphate.

If the mechanism is random, one would therefore expect that citrulline is also competitive towards ornithine. Fig. 13 shows that inhibition by citrulline is noncompetitive. This effect is readily explained by assuming that an abortive complex of the type

J.-P. Simon and V. Stalon 579

[Citrulline] (rnM)

0.1 [Carbarnoylphosphate]-’ (kM-’)

3 I

[Citrulline] (mM)

Fig. 12. Product inhibition of the forward reaction by citrulline. (A) Reciprocal reverse velocity versus reciprocal carbamoylphos- phate concentration. Ornithine concentrations were taken equal to 200 pM. Citrulline concentrations are shown. u is the relative velocity. (B) Replot of slopes against citrulline concentration

enzyme . ornithine . citrulline (EBP) may be formed in this system. However, such a complex seems unlikely. Here again, the results show that the Ki slope of the competitive inhibition of citrulline towards carbamoylphosphate is constant under the conditions investigated (when ornithine was nonsaturating and above the Michaelis constant) (Table 3).

CONCLUSIONS

The arsenolytic cleavage of citrulline shows un- ambiguously that the mechanism is of the rapid- equilibrium random type with concomittant formation of enzyme . citrulline . phosphate and enzyme . orni- thine . arsenate dead-end complexes.

That the mechanism is also random in the direc- tion of citrulline synthesis is based on the following facts : (a) phosphonoacetate, competitive with respect to carbamoylphosphate, is noncompetitive with re- spect to ornithine, while 2,4-diaminobutyrate, com- petitive with respect to ornithine, is noncompetitive

- C

c a a,

c $ c CI

! / 100

[Citrulline] (rnM)

[Citrull ine](rnM)

I00

1 50 . -

0

I

10 [Ornithine]-’ (mM-’)

0

100 [Citrulline] (mM)

Fig. 13. Product inhibition of the forward reaction by citrulline. (A) Reciprocal reverse velocity versus reciprocal ornithine concen- tration. Carbamoylphosphate concentrations were taken equal to 6.5 pM. Citrulline concentrations are shown. L’ is the relative velo- city. (B) Replot of slope against citrulline concentration. (C) Replot of the intercepts versus citrulline concentrations

with respect to carbamoylphosphate: (b) phosphate induces ornithine inhibition which is competitive with respect to carbamoylphosphate.

Thus, phosphate, citrulline, ornithine, carbamoyl- phosphate and dead-end inhibitors such as phosphono- acetate and 2,4-diaminobutyrate all appear to combine with the free enzyme. The values of the inhibition constant determined for ornithine and phosphono- acetate in both directions are in good agreement (Tables 1 and 4).

It is still necessary to explain the discrepancy found in the constants for phosphate and citrulline as well as the effect of norvaline. If the system is random for ornithine and carbamoylphosphate, the norvaline combines only with the binary enzyme . carbamoyl- phosphate complex. Multiple inhibition studies show that the binding of phosphate does not exclude ornithine and citrulline binding but that the binding of phosphate and the binding of norvaline are mutually exclusive. In the range of concentration of its dissociation constant (0.2 mM) determined in the

580 Steady-State Kinetic Analysis of L-Ornithine Carbamoyltransferase

Table 4. Calculated values ojthe product anddead-endconstants,for the reaction in the forward direction The reaction was assumed to have either an ordered mechanism with two dead-end complexes EP and EBQ, or rapid equilibrium with three dead-end complexes EQ, EBQ and EBP. Calculations were made substituting into the given relationships between true and apparent con- stants the values for the apparent constant (Table 3), the value for K,, Ki,, Kb and Kib (Table 2) and the fixed substrate concentrations given in the table. The true values for the inhibition constant were calculated by assuming that the rate equation for the reaction is represented by the equation t' = V [A] [B]/(K,, Kb + K, [B] + Kb [A] + [A] [B]). In the presence of phosphate, citrulline or phosphonoacetate, the rate equation is given by the above equation with the K,, Kb and K, terms multiplied by (1 + [I]/Ki) in ordered mechanism, while the K,. Kh and K, terms are multiplied the factor (1 + [I]/Ki) and (1 + [I]/Kl) respectively in random mechanism. In the presence of norvaline the Kb term is multiplied by (1 + [I]/Ki) for both mechanism. In the presence of 2,4-diaminobutyrate the Ki, Kb and Kb terms are multiplied by the factors (1 + [I]/Ki) and (1 + [I]/Kr) respectively in random mechanism, while the Ki, K b and Kb terms are multiplied by (1 + [I]/Ki)

Inhibitor Variable substrate Fixed Relationships between true and apparent constant - ~ - - substrate

ordered mechanism random mechanism

Phosphate (Q)

Citrulline (P)

_ _ -~

Norvaline

Phosphonoacetate

2,4-Diaminobutyrate

carbamoylphosphate (A)

ornithine (B)

-

carbamoylphosphate (A)

ornithine (B)

~ ~~

carbamoylphosphate (A)

ornithine (B)

- ~- ~

carbamoylphosphate (A)

ornithine (B)

mM

0.2

2.5

0.00654

0.2

40

0.00654

ornithine (B)

carbamoylphosphate (A)

0 2

0 00654

2 5

0 2

2

0 00654

~~ ~

0 21

0 00654

Ki, = Ki, = 2.75

Ki, = Kj, = 2.2

Kip = K,s = 30

same as ordered mechanism

Kip same as ordered mechanism

same as ordered mechanism

same as ordered mechanism

. ._

same as ordered mechanism

J.-P. Simon and V. Stalon 581

forward direction, norvaline is not inhibitory in the reverse reaction, the arsenolytic cleavage of citrulline.

Although the mechanism appears random, the competitive inhibitions of phosphate, citrulline and phosphonoacetate are more easily explained by an ordered mechanism. Indeed according to Cleland, ordered and random mechanisms can be distinguished by determing Ki slopes for the inhibitors at different concentrations of the non-varied substrate, ornithine. A constant Ki slope indicates an ordered mechanism and a changing Ki slope indicates a random mecha- nism. The results show the Ki slope to be constant under the conditions investigated, irrespective of the concentration of ornithine (see Table 3). These results are more consistent with an ordered mechanism. In a random mechanism, if the majority of the reaction flux, when both substrates are present at their K,,, levels, goes through the path with carbamoylphos- phate adding before ornithine, then the pattern for inhibition is like that of an ordered mechanism. This situation has been encountered for yeast hexokinase [ l l ] and alcohol dehydrogenase [12,13].

Thus, if carbamoylphosphate is preferably linked first, i t is apparent that competitive inhibition with respect to carbamoylphosphate requires product in- hibition to be noncompetitive with respect to orni- thine. It is no longer necessary to postulate the existence of an unlikely enzyme . ornithine . citrulline ternary complex.

On the basis of this work, the kinetic mechanism for the ornithine carbamoyltransferase of Succhuvo- myces cevevisiue thus appears to be of the rapid- equilibrium random type for the arsenolytic cleavage of citrulline. In the forward direction, although the

mechanism is also random, most of the data must be explained by a preferred pathway with carba- moylphosphate adding first.

How the epiarginasic regulation of the ornithine carbamoyltransferase system operates is now well understood and will be published as soon as possible.

This work was supported by grant 2/4245/75 from the F0nd.s de la Recherche Fondamentale Collective. We are grateful to Dr M. Penninckx for supplying phosphonoacetate and to K. Broman for reading the manuscript.

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