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Eur. J. Riochem. 58, 533-538 (1975) Interaction between Arginase and L-Ornithine Carbamoyltransferase in Saccharomyces cerevisiae The Regulatory Sites of Arginase Michel PENNINCKX Laboratoire de Microbiologie, Universite Libre de Bruxelles, et lnstitut dc Recherches du Centre d'Enseignement et de Recherches des Industries Alimentaires et Chimiques, Bruxelles (Received May 31/July 31, 1975) The inhibition of ornithine carbamoyltransferase by arginase in Saccharomyces cerevisiae, which is under the control or arginine and ornithine, involves a regulatory site for arginine on the arginase distinct from its catalytic site. This regulatory site is responsible for the reinforcement effect of arginine on the inhibition of ornithine carbamoyltransferase by arginase. The binding site of ornithine carba- moyltransferase on arginase is also shown by our analysis. Inhibition of L-ornithine carbamoyltransferase of Saccharomyces cerevisiae by its own arginase is now understood at the level of the quaternary structure of the enzymes. These have a common trimeric quaternary structure and, thus, the 1 : 1 regulatory complex they form is most probably an hexamer [l]. Arginine and ornithine, respectively substrates of arginase and ornithine carbamoyltransferase, are specific effectors of this regulation [2-41. It was early shown that inhibition by ornithine results from binding to ornithine carbamoyltransferase at a regula- tory site distinct from the catalytic one [3]. Inhibition of ornithine carbamoyltransferase by arginase is rein- forced by addition of L-arginine [2]. The question of the site of arginine inhibition remained open. This site appears to be restricted to arginase on the basis that arginine has no effect on ornithine carbamoyl- transferase [3]. We also know a mutant from arginase devoid of catalytic power, which can inhibit ornithine carbamoyltransferase; maximal inhibition occurring in the presence of L-arginase is the case with the wild-type form [2]. This suggests a separation between catalytic and regulatory functions of arginine, which could be exercised by topographically distinct sites. Moreover the regulatory effect of arginine is complete at 0.1 mM, which is a concentration well below the catalytic K, or arginine [5]. We present here more direct evidence for the existence of distinct catalytic Enzymes. Arginase or L-arginineamidinohydrolase (EC 3.5.3.1); r-ornithine carbamoyltransferase (EC 2.1.3.3); urease or urea amidohydrolase (EC 3.5.1.5) ; r-chymotrypsin or chymotrypsin A (EC 3.4.21.1); carboxypeptidase A or carboxypolypeptidase (EC 3.4.12.2). -~ and regulatory sites of arginine on arginase. Our analysis is based on the action of analogues, affinity labelling and desensitization by mild proteolysis. The binding site of ornithine carbamoyltransferase on arginase is also shown by mild proteolysis. MATERIALS AND METHODS Strains and Cultures Strain MG409, source of ornithine carbamoyl- transferase, and strain 12430c, source of arginase, were previously described as well as the respective culture conditions [l]. Partial Purification of Ornithine Carbamoyltransferase Ornithine carbamoyltransferase was partially puri- fied by the two initial steps of the five-step published procedure [l]. The final dialysis buffer is replaced by 50 mM Tris maleate pH 8. The enzyme solution is stored at - 19 "C without loss of activity. Partial Purfication of Arginase Arginase was purified by a five-step procedure including the four first steps and the sixth step of our published procedure [l]. The arginase used in the inhibition assay was to be more extensively purified to eliminate contaminating ornithine carbamoyltrans- ferase always present in small amounts in yeast cells growing on arginine as nitrogen source. The final enzyme solution was dialyzed against 50 mM Tris-

Interaction between Arginase and l-Ornithine Carbamoyltransferase in Saccharomyces cerevisiae : The Regulatory Sites of Arginase

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Page 1: Interaction between Arginase and l-Ornithine Carbamoyltransferase in Saccharomyces cerevisiae : The Regulatory Sites of Arginase

Eur. J . Riochem. 58, 533-538 (1975)

Interaction between Arginase and L-Ornithine Carbamoyltransferase in Saccharomyces cerevisiae The Regulatory Sites of Arginase

Michel PENNINCKX

Laboratoire de Microbiologie, Universite Libre de Bruxelles, et lnstitut dc Recherches du Centre d'Enseignement et de Recherches des Industries Alimentaires et Chimiques, Bruxelles

(Received May 31/July 31, 1975)

The inhibition of ornithine carbamoyltransferase by arginase in Saccharomyces cerevisiae, which is under the control or arginine and ornithine, involves a regulatory site for arginine on the arginase distinct from its catalytic site. This regulatory site is responsible for the reinforcement effect of arginine on the inhibition of ornithine carbamoyltransferase by arginase. The binding site of ornithine carba- moyltransferase on arginase is also shown by our analysis.

Inhibition of L-ornithine carbamoyltransferase of Saccharomyces cerevisiae by its own arginase is now understood at the level of the quaternary structure of the enzymes. These have a common trimeric quaternary structure and, thus, the 1 : 1 regulatory complex they form is most probably an hexamer [l]. Arginine and ornithine, respectively substrates of arginase and ornithine carbamoyltransferase, are specific effectors of this regulation [2-41. It was early shown that inhibition by ornithine results from binding to ornithine carbamoyltransferase at a regula- tory site distinct from the catalytic one [3]. Inhibition of ornithine carbamoyltransferase by arginase is rein- forced by addition of L-arginine [2]. The question of the site of arginine inhibition remained open. This site appears to be restricted to arginase on the basis that arginine has no effect on ornithine carbamoyl- transferase [3]. We also know a mutant from arginase devoid of catalytic power, which can inhibit ornithine carbamoyltransferase; maximal inhibition occurring in the presence of L-arginase is the case with the wild-type form [2]. This suggests a separation between catalytic and regulatory functions of arginine, which could be exercised by topographically distinct sites. Moreover the regulatory effect of arginine is complete at 0.1 mM, which is a concentration well below the catalytic K , or arginine [5]. We present here more direct evidence for the existence of distinct catalytic

Enzymes. Arginase or L-arginine amidinohydrolase (EC 3.5.3.1); r-ornithine carbamoyltransferase (EC 2.1.3.3); urease or urea amidohydrolase (EC 3.5.1.5) ; r-chymotrypsin or chymotrypsin A (EC 3.4.21.1); carboxypeptidase A or carboxypolypeptidase (EC 3.4.12.2).

-~

and regulatory sites of arginine on arginase. Our analysis is based on the action of analogues, affinity labelling and desensitization by mild proteolysis. The binding site of ornithine carbamoyltransferase on arginase is also shown by mild proteolysis.

MATERIALS AND METHODS

Strains and Cultures

Strain MG409, source of ornithine carbamoyl- transferase, and strain 12430c, source of arginase, were previously described as well as the respective culture conditions [l].

Partial Purification of Ornithine Carbamoyltransferase

Ornithine carbamoyltransferase was partially puri- fied by the two initial steps of the five-step published procedure [l]. The final dialysis buffer is replaced by 50 mM Tris maleate pH 8. The enzyme solution is stored at - 19 "C without loss of activity.

Partial Purfication of Arginase

Arginase was purified by a five-step procedure including the four first steps and the sixth step of our published procedure [l]. The arginase used in the inhibition assay was to be more extensively purified to eliminate contaminating ornithine carbamoyltrans- ferase always present in small amounts in yeast cells growing on arginine as nitrogen source. The final enzyme solution was dialyzed against 50 mM Tris-

Page 2: Interaction between Arginase and l-Ornithine Carbamoyltransferase in Saccharomyces cerevisiae : The Regulatory Sites of Arginase

534 Arginine in Ornithine Carbamoyltransferase-Arginase Interaction

HC1 pH 8 buffer containing 1 mM arginine. The enzyme solution is stored at - 19°C without loss of activity. Before use the solution is dialyzed against 50 mM Tris-HC1 pH 8 buffer.

Other Enzymes and Proteins

Urease (Sigma type 111) is added in the ornithine carbamoyltransferase assay to destroy urea when inhibition by arginase in the presence of arginine is investigated.

Bovine serum albumin (Sigma) is used as standard for protein determination. Casein according to Ham- merstein (Merck) is used for control of activity of the commercial proteolytic enzymes used in the work. Bovine pancreas a-chymotrypsin is a Boehringer (Mannheim) product. Bovine pancreas carboxypep- tidase A is a Worthington product.

Enzyme Assays

a-Chymotrypsin and carboxypeptidase A are as- sayed by the Kunitz method [6 ] . It has been checked that the effectors, arginine and agmatine (see Results), have no influence on the activity of the two pro- teolytic enzymes.

Ornithine Carbamoyltransferase Activity

Ornithine carbamoyltransferase activity determi- nation is adapted from Jones et al. [7]. Citrulline formed was measured by the colorimetric method of Archibald [8]. The method is one previously de- scribed [3]. Units of activity of ornithine carbamoyl- transferase at pH 8.0 are expressed in pmol of citrulline produced/h at 15 "C in the presence of 7 mM orni- thine and 10 mM carbamoylphosphate as substrates.

Arginase Activity

Arginase is assayed without the previously men- tioned activation step with MnCl, [3]. To 1 ml of an enzyme solution in 100 mM Tris-HC1 buffer, pH 8, is added 1 ml of an L-arginine-HC1 solution at the desired concentration. After 30 min incubation the reaction is stopped by addition of 2 ml 1 M HCl. Urea is determined on the whole volume by the colorimetric method of Archibald [9].

Alkjjlution of Arginase

The alkylation of arginase by different amounts of ~-2-amino-4-oxo-5-chloropentanoic acid was per- formed in 50 mM Tris-HC1 buffer, pH 8, at 30 "C. The final protein concentration of the partially purified arginase extract in the presence of the chemical is 0.5 mg/ml. Aliquots of 5 pg and 0.5 pg of proteins

were tested for the action of the chemical on the catalytic and the regulatory activities of arginase (see Results for details of activity measurements).

Proteolysis of Arginase by a-Chymotrypsin and Carboxypeptidase A

Proteolysis of arginase was performed in 50 m M Tris-HC1 buffer pH 8. Final concentrations of chymo- trypsin and carboxypeptidase A were respectively 10 pg/ml and 70 pg/ml. Proteolysis was stopped by twenty-fold dilution in 100 mM Tris-HC1 buffer pH 8. It has been checked that the final concentration level of the proteolytic enzyme after dilution does not affect in any way the catalytic and regulatory proper- ties or arginase and ornithine carbamoyltransferase.

Other Procedures

Protein was determined quantitatively by the Folin-phenol method [lo] with bovine serum albumin as standard.

Chemicals

Tris buffer, L-ornithine-HC1, L-arginine-HC1, dili- thium carbamoylphosphate and cT-aminovaleric acid were purchased from Sigma, y-guanidinobutyric acid was obtained from Calbiochem and agmatine sulfate from K and K . ~-2-Amino-4-oxo-5-chloropentanoic acid was a gift from Miss B. Schroter. All other chemicals were of reagent grade quality.

RESULTS

THE REGULATORY SITE FOR ARGININE ON ARGINASE

Competition with Ornithine and Effect of Ornithine on the Regulatory Function of Arginine

Ornithine, substrate of ornithine carbamoyltrans- ferase and product of arginase action, is also known to be a classical competitive inhibitor of arginase with respect to arginine from many organisms [l 1 - 131. It was of special interest to test it on the S. cerevisiae enzyme because in our classical assay of ornithine carbamoyltransferase inhibition by arginase, ornithine which is substrate and effector of ornithine carbamoyl- transferase, is present at 15 mM concentration and maximal reinforcement effect of the inhibition of ornithine carbamoyltransferase by arginase is pro- duced by 0.1 mM arginine [5].

Fig.1 shows that ornithine is a competitive in- hibitor of the catalytic power of arginase, with respect to arginine, and that incubation in the pres-

Page 3: Interaction between Arginase and l-Ornithine Carbamoyltransferase in Saccharomyces cerevisiae : The Regulatory Sites of Arginase

M . Penninckx 535

20

15

5 10

5

0 0.1 0.3 0.5 0.7 l / [ A r g i n i n e ] (mM- ' )

Fig. I . Competitive inhibition of arginase wiih respect to arginine by r-ornithinr. 4 pg of protein from a partially purified arginase extract is incubated at 15°C (0) without L-ornithine; with (A) 1 mM L-ornithine; (0) 3 mM L-ornithine; (0) 5 mM L-ornithine; ( x ) 10 mM L-ornithine and with different amounts of L-arginine. I' is the velocity in pmol urea formed in 30 min. The same experiment performed in the presence of 15 units of ornithine carbamoyltransferase gives exactly the same pattern

ence of a saturating amount of ornithine carbamoyl- transferase does not modify the inhibitory pattern. This confirms a preliminary observation concerning the insensitivity of arginase activity to the binding with ornithine carbamoyltransferase [5]. The Ki value for ornithine is 0.25 mM and K , for arginine is 1 mM.

Fig.2 describes a reciprocal-type plot of the supplementary inhibition of ornithine carbamoyl- transferase by arginase in the presence of various amounts of arginine effector. The inhibitory function of arginine follows a classical Langmuir adsorption isotherm and a dissociation constant (KR) for arginine of 15 pM can be determined. Moreover Fig.2 shows that ornithine does not modify the regulatory dissoci- ation constant of arginine.

It seems thus that steric occupation of the catalytic site of arginase by the competitive inhibitor L-ornithine has no effect on the regulatory properties of arginine.

Competition with b-Aminovalerate and Effect of b-Aminovalerate on the Regulatory Function of Arginine

b-Aminovalerate (the product of a-deamination of ornithine) is a competitive inhibitor of arginase with respect to arginine. The Ki is 100mM. The presence of 200 mM 8-aminovalerate has no effect on the regulatory function of arginine. Moreover a con- centration of 200 mM b-aminovalerate does not inhibit ornithine carbamoyltransferase. This inhibitor, acting on the arginase active, site, does not modify the regulatory function of arginine, and thus cannot replace or interfere with arginine in its regulatory function.

0.10

Q . - 0.05

1O31O4 2.104 5.104 105 I / [ Arginine] (M-')

Fig. 2. Rec@rocal plot of the regulatory ej'kri oj'arginine. 14 pg of protein from a partially purified ornithine carbamoyltransferase extract is incubated at 15 "C in the presence of 0.4 pg of a partially purified arginase extract and with: (0) 5 mM L-ornithine; (0) 15 mM L-ornithine. The concentration of carbamoylphosphate is 10 mM, R is the percentage of supplementary inhibition afforded by various amounts of regulatory arginine relative to the basic level of inhibition produced by arginase alone

Action of Analogues of Arginine

Gudnidinated analogues of arginine, inactive as substrates for arginase and without effect on ornithine carbamoyltransferase, were tested for their action on the catalytic and regulatory properties of arginase. Two typical behaviours are shown.

y-Guanidinobutyrate is a noncompetitive inhibitor of arginase with respect to arginine (Fig. 3). The Ki is 50 mM. Fig.4 shows that this analogues does mimic arginine for the regulatory effect with a K , of 170 pM.

Agmatine, decarboxylated analogue of arginine, is a competitive inhibitor of arginase with respect to arginine. The Ki is 16.5 mM. It has a regulatory function with a K , of 110 pM (Fig.4). Guanidinated analogous do not act only on the catalytic or regulatory function of arginine. Since their determined Ki and K, are dissociations constants with very remotes values, it appears that the best explanation of their action involves two different sets of sites: one set acting on the catalytic power of arginase and the other im- plicated in the regulatory behaviour of the enzyme.

Affinity Labelling of the Catalytic Site of Arginase

~-2-Amino-4-oxo-5-chloropentanoic acid (chloro- ketone I) [14] is a potent reactive analogue of L-orni- thine, competitive inhibitor of arginase with respect to arginine. We observe for arginase time-dependent and dose-dependent pseudo-first-order irreversible inactivation. L-Ornithine protects against this in- activation. Kitz and Wilson treatment [15] of the observed pseudo-first-order inactivation constant

Page 4: Interaction between Arginase and l-Ornithine Carbamoyltransferase in Saccharomyces cerevisiae : The Regulatory Sites of Arginase

536 Arginine in Ornithine Carbamoyltransferase-Arginase lnleraction

I

0 0.1 0.3 0.5 1 .o

Fig. 3. Noncompetitive inhibition of arginasa with rcqwct to urgin- ine by y-guanidinobutyrute. 2.5 pg of protein of a partially purified arginase extract is incubated at 15 "C (0) without y-guanidinobuty- rate; with (A) 10 mg y-guanidinobutyrate; (0) 20 mM g-guanidino- butyrate; (0) 30 inM y-guanidinobutyrate; and with different amounts of L-arginine, u is the velocity in pmol urea formed in 30 min

l /(Arginine] (mM-')

(Fig.5) gives a saturation law characteristic of an affinity label. A dissociation constant (Ki) of 10 mM for the reactive analogue is deduced as well as a dis- sociation constant (Kp) of 1 mM for protective L-or- nithine. This K p of ornithine fits well with the en- zymatically deduced dissociation constant of 1.2 mM for L-ornithine determined in the buffer and temper- ature conditions of the chemical attack (not shown). The catalytic site of arginase appears thus as the target of inactivation.

An 85 %, chloroketone-I-inactivated arginase preparation has 75% of the total inhibitory power of native arginase towards ornithine carbamoyltrans- ferase. The regulatory function of arginine (Fig. 6 ) is completely insensitive to chemical covalent occupa- tion of the active site.

Slight loss of inhibitory capacity of arginase could be explained by some unspecific labelling of the protein. Safe-keeping of the regulatory function of arginine is a good indication of the existence of distinct catalytic and regulatory sites.

Desensitization by Mild Proteolysis

Desensitization has been one of the first methods for showing allosteric interactions [16,17]. Heat de- sensitization of the regulatory function of L-ornithine carbamoyltransferase has shown a clear distinction between catalytic and regulatory behaviour of the effector [3]. This procedure, applied to arginase, has failed to show a clear distinction between catalytic and regulatory functions of L-arginine. We then turned to the method of mild proteolysis. Since the first elegant works of the Carlsberg Laboratory [18] on the degradation of proteins by enzymes, it has appeared that mild proteolysis is a method for showing de-

lo3 lo4 2 .lo4 3.10~ l/[Effector] (M-')

Fig. 4. Rrgulutory aflfrct of 7-guanidinohutyrutc a t i d uginutinc. Same conditions as for Fig.2, but with 5 mM L-ornithine and 10 mM carbamoylphosphate as substrates and substitution of regulatory L-arginine by various amounts of y-guanidinobutyrate (0) and agmatine (0)

sensitization [19] and distribution of distinct sites on proteins [20,21].

Action of a-Chymotrypsin on Arginase

Fig. 7 A shows the action of cr-chymotrypsin on the catalytic and regulatory activities of arginase. It can be deduced that susceptibilities to proteolytic attack of catalytic and regulatory functions of arginase are not the same. Fig. 7 B shows that 1 mM arginine and 1 mM agmatine addition affords a substantial protection against the loss of inhibitory activity without significant effect on the evolution of catalytic activity of arginase. The fact that agmatine acts in a low concentration range characteristic of the regu- latory ability of this analogue (Kr = 110 pM, see above) suggests that the protective mechanism in- volves a binding site remote from the catalytic one (Ki = 16.5 mM). Moreover, protection of arginase by agmatine shows that the site which recognizes the analogue and thus most probably the regulatory site for arginine is located on arginase.

THE BINDING-SITE OF ORNITHINE CARBAMOYLTRANSFERASE ON ARGINASE

Treatment with carboxypeptidase A results in a preparation of arginase 80 catalytically active but devoid of inhibitory power towards ornithine carbamoyltransferase (Fig. 8). The effectors, arginine and agmatine, do not protect against desensitization of arginase. This experiment shows that the carboxy- peptidase-sensitive site is probably implicated in the binding ability of arginase with ornithine carbamoyl- transferase.

Page 5: Interaction between Arginase and l-Ornithine Carbamoyltransferase in Saccharomyces cerevisiae : The Regulatory Sites of Arginase

M. Penninckx 537

0.15

200

150

c . - 103

53

0.10

p. . c

0 0.1 0.2 0.5 1 .o 103104 2.104 5 . 1 0 4 I o5 l/[Chloroketone I] (rnM-') l / [Arginine] (M-')

Fig. 5. A[f;nity lnhelling of' the cutulj,tic site o/ wgirinse b!, drluro- ketone I und proredion by L-ornithine. See Materials and Methods for the alkylation conditions. Ki values are the pseudo-first-order inactivation constants (min-') calculated for the different amounts of the chemical: (0) without protection; (0) with 1 mM L-ornithine. Arginase activity was tcstcd in the presence of 20 mM L-arginine at 15°C

Fig. 6 . Effect of' chloroketone I inactiwtion of urginusr on (lie regulutory funcrion of L-arginine. Same conditions as for Fig. 2 except 0.5 pg of protein of a partially purified arginase extract; 5 mM L-ornithinc and incubation temperature at 20 'T. (0) Native arginase; (0) 85% inactivated arginase

I

0 10 30 60 0 I0 30 60 Time (min) Time (min)

Fig. 7. Action oj'a-cliwnotr,vpsin on arginase. (A) 0.5 mg/ml of a partially purified arginase extract is incubated in 50 mM Tris-HC1 buffer, pH 8. at 15?C for different times in the presence of 10 pg/ml r*-chymotrypsin. At each time twenty-fold-diluted aliquots (see Materials and Methods) are tested for activity and inhibition of ornithine carbamoyltransfcrasc as already described. (B) Samc conditions as for (A) but protcolysis was performed in the presence of 1 mM agmatine. (0) "4 of activity of arginase: tested in the presence of 20 mM L-arginine as substrate; (0) of the remaining inhibitory power of arginase towards ornithine carbamoyltransferase: assayed in the presence of 1 mM L-arginine, 15 rnM L-ornithinc and 10 mM carbamoylphosphate. Proteolysis performed in the presence of 1 mM L-arginine gives essentially the same pattern as in the presence of 1 m M agmatinc

The catalytic activity of arginase seems independent from the binding-site of ornithine carbamoyltrans- ferase on arginase. Moreover, failure of arginine and agmatine to protect against desensitization is a good indication of a likely physical separation of the regulatory site for arginine and the binding area of ornithine carbamoyltransferase on arginase.

DISCUSSION

Two effectors, ornithine and arginine, are needed for the full expression of the inhibition of yeast

ornithine carbamoyltransferase by the regulatory arginase. It was shown earlier that the regulatory function of ornithine is fulfilled by the presence of a regulatory site on ornithine carbamoyltransferase [3]. As shown here a full parallelism exists for the regulatory function of arginine.

The function is also exerted by means of a topo- graphically distinct site from the catalytic site on arginase. This observation results from a coherent bundle of information gained by essentially chemical means. A clear mutational event resulting in a catalytically inactive but fully regulated arginase was

Page 6: Interaction between Arginase and l-Ornithine Carbamoyltransferase in Saccharomyces cerevisiae : The Regulatory Sites of Arginase

538 M. Penninckx : Arginine in Ornithine Carbamoyltransferase-Arginase Interaction

." - I

0 10 30 40

Fig. 8. Action of curhos~pc~~~riduse A 017 urginase: the binding site o j ornithine carbamo~~ltran.~era.~e on arginase. 0.5 mg/ml of a par- tially purified arginase extract is incubated in 50 mM Tris-HC1 bufl'er, pH 8.0, at 15 "C for din'erent times in the presence of 70 pg/ml carboxypeptidase A. At each time twenty-fold-diluted aliquots (see Materials and Methods) are assayed for activity and inhibition of ornithine carbamoyltransferase as described in the legend of Fig. 7. Proteolysis performed respectively in the presence of 1 mM L-arginine and 1 mM agmatine gives the same pattern as in the absence of effectors. (0) Catalytic activity; (O), inhibitory power

Time (rnin)

reported earlier [ 2 ] . Nevertheless this information is only suggestive of the existence of distinct catalytic and regulatory sites because the described mutation may have only resulted in modification of an amino acid implicated in the catalytic process and not in the binding of the substrate. This result shows that arginase need not to be necessarily catalytically active for the expression of its regulatory power. The binding site of ornithine carbamoyltransferase on arginase is also shown in our investigations. The distribution of surface sites on arginase appears as follows : catalytic centers working freely and a binding area for the regulatory target, ornithine carbamoyl- transferase. This binding area is partially under the control of separate regulatory centers.

It must be said that during the completion of our work a paper mentioning a similar system of inhibition of ornithine carbamoyltransferase by arginase in the procaryotic genus Bucillus has appeared [22]. Orni- thine and arginine are also effectors of this regulation and the authors have some partial indications that arginine could act by means of a regulatory site.

Inhibition of ornithine carbamoyltransferase by regulatory arginase prevents the occurence of a waste- ful urea cycle which could operate when arginine is catabolized [2,4]. This control mechanism involves the integrity of the regulatory site of ornithine [3,4].

In our opinion the physiological meaning of a regula- tory site for arginine must be sought in a reverse metabolic situation where biosynthesis of arginine is needed. It can be thought that the creation of anabolic conditions is quickly followed by a consequent de- crease of the cytoplasmic pool of arginine. Full syn- thesis of anabolic ornithine carbamoyltransferase is rapidly achieved. Until dilution of now unnecessary arginase by cellular division, a potent wasteful urea cycle is possible because of the activity of ornithine carbamoyltransferase. Thanks to the enzymotropic regulation of the enzyme and the high affinity of the regulatory site for arginine an efficient control could be achieved.

I thank Professor J.-M. Wiame and Dr V. Stalon for helpful advice and K. Broman for help with the English. This work was supported by contract no. 985 from the Fonds de la Recherrhc Fondamentale Collr>tive.

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M. Pcnninckx, Institut dc Recherches du C.E.R.I.A., 1 Avenue Emile-Gryzon, B-1070 Bruxelles, Belgium