An aspartate transcarbamylase lacking catalytic subunit interactions

J. Mol. Biol. (1973) 78, 687-702

An Aspartate Transcarbamylase Lacking Catalytic Subunit Interactions 1I.t Regulatory Subunits are Responsible for the Lack of Co-operative Interactions between Catalytic Sites. Drastic Feedback Inhibition does not Restore these Interactions D.LNI&LE KERBIFUOU AND GUY HERVI?

Service de Biochimie du Dbpartement de Bdogie, CEN- SACLA Y Commissariat d I’Energie Atmique, B.P. no. 2 91190 Gif-sur- Yvette, France (Received 21 February 1973) It has been previously reported (Kerbiriou & Herve, 1972) that, when a uracilrequiring mutant of Eacherichicr coli is derepressed for the biosynthesis of the enzymes of the pyrimidine pathway in the presence of 2-thiouracil, it synthesizes a modified aspartate transcarbamylase which is still sensitive to the feedback inhibitor CTP, but which does not show homotropic positive interactions between catalytic sites. It is shown here that these homotropic interactions do not reappear upon strong inhibition by CTP, indicating that the two types of interactions are really disconnected and must involve different molecular mechanisms. CTP is acting at the level of the apparent K, of the enzyme for aspartate. It is also the case for ATP, which stimulates 2-thioumcil aspartate-trtmscarbamylase. Kinetic studies of the hybrid molecules made up of subunits prepared from normal and modified enzymes show that it is a modification at the level of the regulatory subunits which is responsible for the lack of co-operative interactions between catalytic sites. These results are discussed in terms of a four-state model.

1. Introduction The structure and the properties of Escherichia coli aspartate transcarbamylase (carbamoylphosphate: L-aspartate carbamoyl-transferase E.C.2.1.3.2.), the first enzyme of the pyrimidine pathway, have been extensively reviewed (Gerhart, 1970; Khschner, 1971; Jacobson & Stark, 1972). This allosteric enzyme is made up of two kinds of subunits: “ catalytic subunits” which possess the catalytic activity but are insensitive to CTP the feedback inhibitor, and “regulatory subunits” which are devoid of any catalytic activity, but bear the sites for binding CTP. Upon mixing under appropriate conditions these two types of subunit recombine to give back a fully active feedback-inhibited native enzyme (Gerhart & Schachman, 1965). The native enzyme exhibits the two kinds of interactions which are characteristic of allosteric enzymes :

(1) homotropic positive interactions as a sigmoidal

relationship

t Paper I in this series is Kerbiriou

between catalytic

between initial & Herd

(1972). 687

velocity

sites, which are visualized and substrate

concentratio

n;

688

D. KERBIRIOU

AND

G. HERVfi

(2) heterotropic interactions between regulatory and catalytic sites, allowing for the inhibition of the catalytic activity upon binding of the feedback inhibitor to the regulatory sites.

It has been previously reported (Kerbiriou & Herve, 1972) that when a uracilrequiring mutant of E. wli is derepressed for the biosynthesis of the pyrimidine pathw&y enzymes in the presence of 2-thiouracil, it synthesizes a modified aspartate transcarbamylase called 2-ThioU-ATCaset. This enzyme, which contains the two kinds of subunits in normal proportion, does not show co-operative interactions between catalytic sites (homotropic interactions), but still possesses the sensitivity to the feedback inhibitor CTP (heterotropic interactions). In the studies described below, it is shown that, even in the presence of high concentrations of CTP which strongly inhibit the modified enzyme, the homotropic positive interactions do not reappear. This result indicates that CTP is not acting by shifting a conformational equilibrium which would be displaced in the opposite direction by aspartate, during homotropic co-operative interactions between catalytic sites. In other words, homotropic and heterotropic interactions are clearly not connected and must correspond to different molecular mechanisms. This interpretation is also supported by the fact that the modified enzyme is stimulated by ATP. In addition, it is shown that the lack of co-operative interactions between catalytic sites in 2-ThioU-ATCase is the result of a modification of the regulatory subunits. This result emphasizes the importance of the regulatory subunit conformation for the transmission of information between catalytic sites.

2. Materials and Methods (a) Chemti Carbamyl phosphate, aspartate, histidine and 2-amino-2-methyl-l,3 propandiol were purchased from Fluka; uraeil from Serlabo; 24hiouracil from Cycle Chemicals; CTP from P.L. Bioohemicals; ATP and bovine serum albumin from Sigma; cacodylic acid from Prolabo and [14C]aspartate from Service de Biochimie, CEN-Saelay, France. (b) Bactwid

&vain

The partially diploid mutant of E. coli used in these experiments (Gerhart & Holoubek, fornia.

1967) wae generously provided

for ATCase production by Dr J. Gerhart, Berkeley, Cali-

(c) Puri&ation of enzymes The mutant was grown in the presence or absence of 2-thiouracil, and 2-ThioU-ATCase was purified as already reported (Kerbiriou & Her&, 1972). Normal ATCase was purified according to Gerhart t Holoubek (1967).

(d) Determination

of enzymk activiiy

(i) Standard conditions In most cases the ATCaae activity of the different preparations wsa determined aa already reported (Kerbiriou & Her&, 1972), in the presence of 5 X 10m2 M-Trig-acetate (pH 8), 2 x lOma aa-aspartate and 5 x lob3 M-carbamyl phosphate. t Abbreviations used: aspartate transoarbamylase

ATCaae: obtained

aspsstate transcarbamylase; in the presence of .Z-thiouracil.

2-ThioU-ATCase,

modified

MODIFIED

ASPARTATE

089

TRANSCARBAMYLASE

(ii) Enzymic unit One unit of ATCase is defined as that amount which catalyses the formation rmol of carbamyl aspartate/h under the standard conditions.

of one

(iii) Assay for atimubtion by ATP The stimulation of ATCase or 2-ThioU-AT&se was tested under the standard conditions in the presence of 5x 10M3 ~-asps&ate, 5x 10e2 M-cacodylate buffer, pH 7, and with or without ATP as indicated. (iv) Ratio of &i&ties at pH 7 and 8.4, and CTP feedbackinhibit&m These determinations were done as already reported (Kerbiriou & Herve, 1972). of the kinetic c&a All the Michaelis-Menten type data were analysed by the Cleland’s iterative (Cleland, 1967), using a Wang 370 computer, with the help of R. Alazard.

(v) Treatment

method

(e) Zinc d&rminatiom The zinc content of the different enzyme preparations wss determined using a Perkin Elmer 290B Atomic Absorption Spectrophotometer. The samples were previously dialysed extensively 3 times against either 10-e M-Tris-acetate buffer, pH 7, or 4 x 10-z rd-imidazolacetate, pH 7. Zinc standard solutions were provided by Perkin Elmer. The protein content of the samples wss determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. The results were corrected by taking into account the 15% higher value given by bovine serum albumin compared to that of AT&se (Grit&, Kerbiriou & Herve, unpublished observations). (f) Dim~&3tim of ATCaae and Z-ThioU-A TCme into au&nits ATCase and 2-ThioU-ATCase were dissociated with p-hydroxymercuribenzoate, and the catalytic and regulatory subunits obtained were separated according to Gerhart & Holoubek (1967). Concentrated solutions of catalytic subunits coming from both enzymes were obtained and kept in 4x lOma M-potassium phosphate buffer (pH 7), 2 x 10T3 Mfl-mercaptoethanol (Gerhart BE Holoubek, 1967). Concentrated solutions of “zinc-regulatory subunits” coming from both enzymes were prepared according to Nelbach et al. (1972) and kept in 4 x lOma Ivr-T&acetate buffer (pH 8), 2 x 10m3 M-6-mercaptoethanol, lo-* M-zinc acetate. (g) Reumatitz~tion of homologous aubunita alepived from

and heterologoua enzymes from ATCaae and Z-ThioU-ATCase

thy?,isolated

In each case, the reassociation of catalytic and regulatory subunits from the solutions described above was done in the following way: O-5 mg of normal or modified catalytic subunits and 0.5 mg of normal or modified zinc-regulatory subunitg were mixed in a total volume of 1.5 ml in the presence of 2 x low3 M-/?-mercaptoethanol and lo-* ~-zinc acetate and the mixture was incubated overnight at 4°C and then for 20 min at 37°C. The reconstituted enzymes obtained were analysed by electrophoresis on polyacrylamide gels as previously described (Perbsl & Her&, 1972), and tested for their enzymic properties after being diluted 250 times with 4 x lOTa M-potassium phosphate buffer, pH 7. (h) Ele&ophw& on polyaerylamide gels The analysis by electrophoresis in polyacrylamide gels was done as already described (Perbal BEHerve, 1972), using 5 pg of each type of enzyme.

3. Results (a) 2-T?iioU-ATCQse

is not a zinc-dejkient

enzyme

It has been well established that the normal native ATCase molecule contains six atoms of zinc which are involved in regulatory and catalytic subunit association and maintenance of the quaternary structure (Gerhart, 1970; Rosenbusch & Weber, 1971a ;

600

D. KERBIRIOU

AND

G. HERVJ-3

Nelbach et al., 1972). Upon dissociation of the enzyme, the zinc atoms are found to be associated with the regulatory subunits, probably at the level of some sulphydryl groups (Rosenbusch & Weber, 1971b; Nelbach et al., 1972). Since the metal is not an absolute requirement for the binding of CTP by the isolated regulatory subunit (Rosenbusch & Weber, 1971b; Cohlberg et al., 1972), one of the hypotheses which were made to account for the special properties of P-ThioUATCase was that it could be a zinc-deficient enzyme as a consequence of a possible chelation of the metal in the growth medium by 2-thiouracil (Kerbiriou & Herve, 1972). In order to test this possibility, the zinc content of 2-ThioU-ATCase was determined by atomic absorption spectrophotometry, for comparison with normal ATCase. The results obtained: 5.4 f 0.5 atoms per molecule of 2-ThioU-ATCase, compared to 6.1 f O-4 atoms per molecule of normal enzyme, do not show a significant difference between the zinc contents of the two enzymes. The averages and standard deviations were calculated from seven determinations made on two different preparations of each kind of enzyme. The small difference which is found cannot exceed one atom per molecule, and is of the same order of magnitude as the variations which are found in normal ATCase from one preparation to another (Rosenbusch t Weber, 1971a; Nelbach et al., 1972). In addition, the properties of 2-ThioU-ATCase were not changed after incubation for 30 minutes at 37°C in the presence of a large excess of zinc. The same result was obtained when the two types of subunit coming from the modified enzyme were recombined as described in Materials and Methods in the presence of an excess of the metal. On the other hand, when the subunits coming from normal ATCase were recombined in the presence of 500 pg 2-thiouracil/ml, the complete enzyme obtained behaved in a normal way. This observation corroborates the fact already reported (Kerbiriou & He&, 1972) that 2-thiouraoil must be present at the time of the biosynthesis of the enzyme to have a chance to modify its properties. These results indicate that even if 2-ThioU-ATCase contained five atoms of zinc instead of six, this would be only a consequence of a more important structural modification. (b) Co-operative interactions between catalytic sites do not reappear upon CTP feedback inhibition In terms of the two models which have been proposed to account for the behavior of alloateric feedback-inhibited enzymes (Monod et al., 1965; Koshland et al., 1966). it is assumed that substrates and feedback inhibitors are acting by inducing conformational changes toward conformations having a higher or lower affinity for the substrate. Concerning the allosteric properties of 2-ThioU-ATCase, two hypotheses were made in order to explain the lack of co-operative interactions between catalytic sites (Kerbiriou & Her&, 1972): (i) 2-ThioU-ATCase could be an enzyme “frozen” in the conformation, or conformations, having the highest affinity for aspartate, that is to say the conformation B in terms of the “sequential” model (Koshland et al., 1966), or conformation R in terms of the “concerted” model (Monod et al., 1965). This enzyme would be unable to reach the conformation or conformations having the lowest affinity for the substrate.

MODIFIED

ASPARTATE

691

TRANSCARBAMYLASE

(ii) Alternatively, this situation could be reversible ; that is to say that, under feedback inhibitor influence, the enzyme would reach the oonformation or conformations having the lowest afllnity for sspartate. In other words, CTP would induce either a sequential conformational ohange toward the “A” state of the “sequential ” model or a conformational change toward the “T” state of the “concerted” model by shifting an equilibrium characterized by an abnormally low allosteric constant L. In such a case, in the presence of a high conoentration of the feedback inhibitor, the co-operative interactions between oatalytic sites would reappear. Only in the first case, that is to say only if the co-operative interactions do not reappear upon strong feedback inhibition, can it be said that homotropic and heterotropic interactions are distinct phenomena and that they must correspond to different molecular mechanisms. In order to test these different possibilities, the kinetic behaviour of 2-ThioUATCase was investigated in the presence of high concentrations of feedback inhibitor, and in comparison with normal ATCase. These experiments were done at pH 8, the conditions under which the homotropio interactions are best visualized in normal ATCase, and in the presence of 10e2 M-CTP, the concentration which inhibits

I

I 02

Aspartate

cancn ( M x IO 2 1 (0)

I

I

I

I

I

5 IO 05 I 2 Aspartate cancn (M x IO 3 )

I

20

(b)

FIQ. 1. Absence of co-operative interactions between catalytio sites of Z-ThioU-ATCase in the presence of high conoentrations of CTP. (a) Initial velocity as a function of asp&&e oonaentration at pH 8. 0.6 H of 2-ThioU-ATCase (--u-n--), and 0.26 pg of ATCase (-O--O-) were inoubeted under standard conditions, but in the presence of lo- a M-CTP snd increasing amounts of asp&&e (from 7 x lo-* to 2.6 X 10Fa M). (b) Small graph: came experiment but in 6X lOWa aa-caoodylate buffer, pH 7, using 1 cg of 2-ThioU-ATCese, in the presenoe of 3.3 x 10e3 M-CTP (-D-n-), and 0.3 big of ATCase in the presence of 2x 1O-3 M-CTP (-O-O-). Large graph: oorresponding Hill plot; the mmximum velocities were estimated by extrapolation of the Lineweaver-Burk double reciprocal plot.

-I

50

692

D. KERBIRIOU

AND

G. HERVfi

2-ThioU-ATCase and ATCase by 66% and SO%, respectively, in the presence of 5 x 10-3 M-aspartate. The results of such an experiment are shown in Figure l(a), where the initial velocities are plotted against the aspartate concentration. It can be seen clearly that under these conditions the sigmoidicity of the curve corresponding to ATCase is enhanced, as already reported by Gerhart & Pardee (1962). In contrast, 2-ThioU-ATCase still behaves in a typioal Michaelis-Menten way, showing that the presence of such a high concentration of CTP does not provoke the reappearance of co-operative interactions between catalytic sites. In order to determine the coeEcient of interaction between catalytic sites by drawing the Hill plot (Hill, 1913), the same kind of experiment was done at pH 7, conditions where a better approximation of the maximum velocities can be obtained. Figure l(b) shows that the Hill coe&ient varies from 1.2 to 34 in the case of normal ATCase, but is invariably equal to 1 in the case of 2-ThioU-ATCase, indicating an absolute lack of co-operative interactions between catalytic sites. These results show that if CTP is acting by shifting an equilibrium between enzyme conformations having high and low affiities for aspartate, these conformations must be distinct from those involved in co-operative interactions between catalytic sites. (0) Characteristics of CTP inhibition

are the same for ATCase and 2-ThioU-ATCase

Allosteric enzymes show the same high specificity toward feedbaok inhibitors as toward substrates. For this reason, it was very unlikely that CTP would feedbackinhibit 2-ThioU-ATCase and ATCase by somewhat different mechanisms. However, several characteristios of their inhibition by CTP were compared and found to be similar: (i) it has been verified that, like catalytic subunits, 2-thiouracil-catalytic subunits are insensitive to CTP. (ii) Figure 2 shows that the pH-dependence of CTP inhibition is similar for the two enzymes. The lower efficiency observed in the case

SO

0 JLL~,--L-l.-L.-,-i 1.1 1 ! I I1 / 114 6 7 8 9 IO FIG. 2. pH dependenoe of CTP inhibition. ATCase (0.4 erg) end 2-ThioU-A’1Case CO.6ale;) were tasted for CTP inhibition under the conditions described in M&k& md Methods at various pH values. CTP ww md at 6 x 10e3 M. soaaodylata for pH valuea from 0 to 7.6. (-O--O--) and (-n--n--). Thebufferwes6xlO-” and 6 x 10-a aa-2-amino-e-methyl-l,3 propandiol-HCI for pH values from 7.6 to 9.8. (--a-+--) and (--m-m-).

MODIFIED

ASPARTATE

093

TRANSCARBAMYLASE

of 2-ThioU-ATCeae has already been reported (Kerbiriou & Her&, 1972). In both cases the sensitivity to CTP increases slowly from pH 7 to pH 10, and quiokly decrertses toward acidic pH values. (iii) The conditions for reversibility by 8spartate of the feedback inhibition were also tested comparatively for both enzymes. This reversibility has been well established at pH 7 in the case of norm81 ATCase (Gerhart $ Pardee, 1964). However, it has been reported by Weitzman & Wilson (1966) that The at alkaline pH, the CTP feedback inhibition cannot be reversed by 88p8Ihte. influence of asp&ate on the CTP inhibition was investigated for both ATCase and 2-ThioU-ATCase. The results presented in Table 1 show that in accord with the finding of Weitzman & Wilson (1966), aspartate does not reverse CTP inhibition at pH 9 as it does at pH 7 and that, in this regard, 2-ThioU-ATCase behaves exactly like ATCase. TABLE 1

Influence of pH and aapartate concentration on feedback inhibition PH 9

PH 7 Aapartate concentration

6x 10-S

10-2

6 x 10-s

10-a

67

43

77

84

44

26

63

67

(M)

Inhibition of ATCase (%I Inhibition of t-ThioU-ATCase

by CTP

(%)

ATCase and 2-ThioU-AT&se were inoubated under standard conditions, with and without 6 x 10e3 a6-CTP, and in the presenoe of either 6 x lo-* M-o8oodylete buffer (pH 7) or 6 x 10ea MI2-emino-2-methyl-l,3 propandiol-HCl buffer, pH 9. The amounts of enzyme ueed were 0.4 m of ATCase or 0.6 cogof 2-ThioU-ATCaaewhentheincubetioneweredoneinthepresenoeof 6 x 10-3~aap8rtate, and 0.3 M of either one or the other enzyme when the inoubations were done in the presence of 10ea M-asp8rtnte.

Prom this series of results it can be reasonably concluded that CTP is feedbackinhibiting ATCase and .%ThioU-AT&se aocording to the same mechenism. This is confirmed by the fact that the two enzymes give 8n identical circular diohroism difference spectrum upon CTP binding (GriiKn, Kerbiriou & Herve, unpublished observations). (d) CTP is acting at the level of the apparent K,

of 2-ThidJ-AT&se

for wpartate

ATCase is considered to be 8118llosteric enzyme of type K, thet is to say that its different conformations should differ by their apparent Km for substrate. This assumption is based on the observation that the effecters, CTP and ATP affect the sigmoidicity of the curve expressing the relationship between initial veloaity and aspartate concentration, but do not affect the maximum velooity (Gerh8rt & Pardee, 1962). Since 2-ThioU-AT&se does not show co-operative aspartate interactions and behaves in a Michaelis-Menten way, the reciprocal plot of its initial velocity 8g8inst asp8rtate concentration is linear and allows for the graphic determination of it8 apparent K, for aspartate (Kerbiriou & He&, 1972).

694

D. KERBIRIOU

AND

G. HERVfi

The fact that the co-operative interactions do not reappear, even upon strong inhibition by CTP, affords an opportunity to determine directly if CTP is ohanging its maximum velocity or its apparent K, for aspartate. Figure 3 shows the LineweaverBurk double reciprocal plot of 2-ThioU-ATCase activity in the absence and presence of CTP at a final concentration of 5 x 10d4 M, which inhibits this enzyme by 32% in the presence of 5 x 10s3 M-aspartate. One can see an important effect of the presence of CTP on the apparent K, for aspartate which increases from 144 f O-16 x low2 M to 2438 f 0.32 x lOma M in the presence of the feedback inhibitor. A 10% difference in the maximum velocity is also observed. This small variation has been repeatedly found and must be significant, but it appears that CTP is acting essentially by altering the apparent K, for aspartate.

-100

0

100 I/Asportote

200 (M“

300

1

Jh. 3. Effeot of CTP on the apparent K, of 2-ThioU-ATCase for aspart&a. 2-ThioU-ATCase (0.6 pg) was tested under the oonditions desoribed iu Materials and Methods for CTP inhibition, in the presenoe of iuareasing amounts of aspartate, and in the presenoe or absence of CTP. The results are presented according to the Lineweaver-Burk double reciprocal plot. (-O--O---) Control without CTP; (-A-A-) in the presenae of 6x lo-’ M-CTP.

(e) ATP activates 2-T&U-ATCase It has been well established that ATP stimulates the activity of ATCase (Gerhart & Pardee, 1962). The fact that the co-operative interactions do not reappear in 2-ThioU-ATCase upon strong inhibition by CTP indicates that the conformational change responsible for the action of this feedback inhibitor must be distinct from the conformational change involved in co-operative interactions between catalytic sites. Since 2-ThioU-ATCase shows maximum affinity for aspartate whatever the concentration of this substrate, it was of interest to determine if ATP is able to stimulate the activity of the modified enzyme. The effect of low3 M-ATP on the activity of 2-ThioU-ATCase was tested in the preuence of varying concentrations of aspartate. The results presented in Table 2 show that ATP activates 2-ThioU-ATCase as it does ATCase. As a control, it can be seen that, under the same conditions isolated catalytio subunits are slightly inhibited. This must correspond to a non-specific competitive inhibition at the level of the binding site for carbamylphosphate, as already reported in the case of CTP (Weitzman & Wilson, 1966; Porter et al., 1969). It is interesting to note that the

MODIFIED

ASPARTATE

695

TRANSCARBAMYLASE

TABLE 2

Activation of 2-ThioU-ATCase Aapart&e concentration (M)

Ellzynle

Effect of 1O-3 M-ATP (%I

3.3 x 10-a 6.6 x 1O-3

+68

2.ThioU-ATCase

3.3 x 10-a 6.6 x 10-S

t30 $13

Catalytic

5x 10-S

-12

ATCase

AT&se !ncubated

by ATP

subunits

(0.4 m), 2-ThioU-ATCese as described in Materials

(0.8 pg) or catalytic and Methods.

+17

subunits

from AT&se

(0.24 pg) were

efficiency of ATP is lower toward the modified enzyme as is the case in CTP feedback inhibition. The reciprocal plot of the initial velocity against aspartate concentration in the presence and absence of IOV3 M-ATP is shown in Figure 4, where it can be seen that ATP is decreasing theK,of 2-ThioU-ATCase for aspartatefrom 144 f 0.16 x 10e2 M to O-74 f 0.1 x 10m2 M. A decrease of the maximum velocity is also observed. The fact that ATP stimulates the activity of the catalytic sites of the modified ATCase suggests that, like CTP, this effector acts by a mechanism somewhat different from the one involved in the homotropic interactions between these catalytic sites.

-100

I L-=LLLd-11 0 100 I/Aspar?ate

200

300

( M-I)

FIG. 4. Effect of ATP on the activity of 2-ThioU-ATCase. 2-ThioU-ATCase (0.6 pg) was tested under the conditions described in Materiels end Methods for ATP stimulation, in the presence of inoreasing amounts of aqart&e, and with or without ATP. The results are presented moording to the Lineweaver-Burk double reoiprooal plot. (-O-O-) Control without ATP; (-n--n-) in the presence of 10W3 M-ATP.

696

D. KERBIRIOU

AND

G. HERVI?

(f) 2-Thiouracil-regulatory subunits are responsible for the luck of co-operative interactions between catalytic sites in 2-ThioU-ATCase It has been shown by Gerhati $ Schaehman (1965) that, upon treatment with p-hydroxymercuribenzoate ATCase separates into catalytic and regulatory subunits, and that under appropriate conditions these two types of subunit can reassociate to re-form an enzyme which possessesall of its original properties (Gerhart, 1970; Rosenbusch & Weber, 1971b). This recombination requires the presence of zinc, which is involved in subunit association (Gerhart, 1970; Rosenbusch & Weber, 197&b; Nelbath et al., 1972). During this process, the formation of a small amount of a higher aggregate has been noticed (Gerhart & Schachman, 1965; Rosenbusch & Weber, 1971b ; Cohlberg et al., 1972), and it has been verified that this aggregate behaves like the reconstituted normal enzyme (Nelbaoh et al., 1972). 2-ThioU-ATCase was dissociated in the same way into so-called 2-thiouracil-catalytic subunits and 2-thiouracil-regulatory subunits. When these two types of subunit were recombined under the conditions described in Materials and Methods, a native enzyme having all the properties of 2-ThioU-ATCase was obtained. As in the case of normal ATCase, the sensitivity to CTP of the reconstituted enzyme was significantly lower than it was before dissociation. This is shown in Table 3, where it can be seen that, in the presence of 5 X 10e3 1\1-aspartate, 5 X lob4 M-CTP inhibits normal ATCase by 52% & 3% before dissociation and reassociation, and by 50% & 8% after; under the same conditions 2-ThioU-ATCase is inhibited by 34% f 3% before dissociation and reassociation, and by 24% f 6% afterwards. TABLE

3

RehaviouT of the hybrid.s 7nade of subuniti prepared from normd and mudi$ed ATCase Type of enzyme

Undiasooieted Un&~~ti Reconstituted Reoonatituted 2-ThioU-Cat. 2.ThioU-Reg.

ATCase 2-ThioU-ATCase ATCase 2-ThioU-ATCase plus norm81 Reg. plus IlOl’ID81 Cat.

Ratio of activities pH 7:pH 8.4 2*36&0.26 0*98&0.06 2.01 f 0.29 0+33*0.14 1.4QO.46 oG33*0.17

o/0 Inhibition by 6x 1O-4 ran-CTP 62*3 34&3 60*8 2456 48+8 21-&7

0.4 118 of undissooieted ATceSe, 0.6 pg of undissooisted ~-T~~oU-ATC~WI and 0.57 pg of each of the following preparations: reoonstituted ATCaee, reconstituted 2-ThioU-ATCese, hybrid enzyme made up of 2-thiouraoil-oatalytio subunits and normal regd8tOIy, hybrid enzyme made up of 2-thioumoil-regulatory subunita and normal oatalytio subunits, were inoubated &8 desoribed in Mat&& and Methods. Avereges and standard deviations were oaloulated from 6 to 8 determindions made on two d&rent preperetions of eaoh hybrid.

Regarding the special properties of 2-ThioU-ATCase, it was interesting to determine which of the catalytic or regulatory subunita are responsible for the loss of cooperative interactions between catalytic sites. In order to answer this question, hybrid molecules made up of normal and 2-thiouracil-modified subunits were prepared. At the same time, as a control, ATCase and 2-ThioU-ATCase were prepared under the same conditions from their isolated subunits. The effeotiveness of the recombination was verified by electrophoresis of the reassociated samples on polyacrylamide gels

I- (b)

/

FIG. 5. Dependenoe of initial velocity on espertate concentration of the reconstituted norm81 and 2-ThioU-ATCam and the oorreaponding hybrids. The reconstituted ATCase (0.3 e), the reoomtituted 2-ThioU-ATCase (O-6 pg), the hybrid enzyme made up of normal oetalytio subunits end 2-thiournoil-reguletory subunits (O-3 pg) and the hybrid enzyme made of 2-ttiOWW3ti-O8td,'tiO subunits and norm&l regulatory aubunita (0.7 ~8) were tested for ATCam aotivity under the standmrd oonditions deeoribed in &f8tEhdf3 and Methods in the preeenoe of inore~ emounte of espmtete (from 7 x lo-’ to 2.6 x lo-’ M). The smell grephu directly represent the intluenee of the eqartata ooneentration on the initial velocities expreaead in ~olea of [WYJoerbamyl aapertata formed in the etanderd oonditions per mg of c&ulytio subunits contained by the hybrids or reoonetituted enzymes. The larger grsphs represent the oormaponding Lineweaver-Burk reciprocal plot. Cat., cetalytio subunits; Reg., regulatory subunits; 2-ThioU-Ueb., eatalytio eubunita coming from 2.ThioWATCese; 2.Thio-U Reg., regulatory subunits from 2-ThioU-ATCaae.

698

D.

KERBIRIOU

AND

G. HERV6

under conditions previously described (Perbal & Herv&, 1972). The results of these analyses are shown in Plate I. In each case, besides the main band of reassociated enzyme, a very small percentage of a more aggregated material was found. These different hybrids were then tested for the characteristics which differentiate between normal and 2-ThioU-ATCase (pH dependence, sensitivity to CTP). Table 3 shows the ratio activities at pH 7 and pH 8.4 and the percentage of feedback inhibition by 5 x 10m4M-CTP of these different molecular species in comparison with undisso&ted native ATCase s,nd 2-ThioU-ATCase. Taking into account the effect of dissociation and reassociation on pH-dependence and sensitivity to CTP which has already been reported, it can be seen that the hybrid composed of 2-thiouracil-catalytic subunits and normal regulatory subunits behaves like a normal reconstituted ATCase ; on the contrary, the hybrid made up of normal catalytic subunits and 2-thiouracilregulatory subunits behaves like a reconstituted 2-ThioU-ATCase. These two hybrids were also tested for the existence or absence of co-operative interactions between catalytic sites in comparison with reconstituted ATCase and 2-ThioU-ATCase by studying the dependence of initial velocity on asp&ate concentration. Figure 5 shows the results of this determination and the corresponding Lineweaver-Burk double reciprocal plots. The following results are obtained. (i) Reconstituted normal AT&se tions (Fig. 5(a)).

still shows the homotropic aspartate interac-

(ii) Reconstituted 2-ThioU-ATCase interactions (Fig. 5(b)).

still does not show the homotropic aspartate

(iii) Of the two hybrids, only the one made up of 2-thiouracil catalytic subunits and normal regulatory subunits shows these homotropic interactions. The hybrid made up of normal catalytic subunits and 2-thionracil-regulatory subunits behaves in a Michaelis-Menten way like native 2-ThioU-ATCase (Fig. 5(a) and (b)). These results together with the data reported in Table 3 show that it is a modification at the level of the regulatory subunits which is responsible for the lack of homotropic co-operative interactions between catalytic sites in 2-ThioU-AT&se. It is, therefore, interesting to note that a precise conformation of the regulatory subunit is more important for the existence of interactions between catalytic sites than for the existence of antagonistic interactions between catalytic and regulatory sites, in which they are directly involved.

4. Discussion ATCase shows the two kinds of interactions which are characteristic of allosteric enzymes : Basically, two kinds of models have been proposed to account for the co-existenoe of these two types of allosteric interactions. (i) The “concerted” or “two state” model (Monod et al., 1965), which postulates that in the absence of any ligand these enzymes present an equilibrium between conformational states differing by their s#inity for the ligands. These ligands, substrates and effeotors are acting by shifting this conformetional equilibrium.

PLATE I. Analysis by gel elwxrophoresis of aspartatc transcarbamylases reconstituted frollr normal and 2.thiouracil subunks. ATCase, 2.ThioU-ATCase and the hybrid molecules made up of subunits coming from normal and 2.thiouracil-modified enzyme, were prepared and analysod by electrophoresis on polyacrylamide gels as described in Materials and Methods. 5 pg of each protein were used. Gel 1. untlisaw ciatod ATCase and catalytic subunits; gel 2. reconst,ituted ATCase; gel 3. enzyme made ,~p of normal catalytic subunits and 2.thiouracil-regulatory subunits; go1 4. enzyme made up of L’thiouracil-catalytic subunits and normal regulatory subunits; gel 5. reconstituted 2-‘l’hioTYAT&se; gel 6. undissociated 2.ThioU-ATCwe and L’-thiouracil-catalytic: subunits; gel 7. rvg~~lat,ory subunits. .\n excess of regulatory subunits can be seen in grls 2 an(l 4.

MODIFIED

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TRANSCARBAMYLASE

699

The conformational change is concerted, which means that in each molecule of enzyme this conformational change affects identically and simultaneously all the subunits. (ii) The “sequential” model (Koshland et al., 1966), established on the basis of the induced-fit theory, postulates that in the absence of any ligand the enzyme is in a conformational state having a given affinity for the substrate. Upon binding of this substrate each subunit would undergo a conformational change affecting its interactions with the neighbouring subunits. As a consequence, these subunits would then bind the substrate more or less easily. In this way the whole enzyme would progressively alter its affinity for ligands. Most of the kinetic data obtained during the past few years have been analysed in an attempt to interpret them in terms of one or the other of these two models. The early data dealing with the ligand-induced conformational changes tested on the basis of reactivity of the sulfhydryl groups toward p-hydroxymercuribenzoate and of a detectable change in sedimentation coescient (Changeux et al., 1968 ; Gerhart & Sohachman, 1968) could be interpreted as fitting the “concerted ” model (Changeux $ Rubin, 1968). The analysis of the relaxation effects, obtained upon temperature or concentration jump experiments carried out in the presence of either succinate or CTP lead to the same conclusion (Eckfeldt et al., 1970; Hammes & Wu, 1971a,b). On the other hand, the effects of both ligands on the antigenicity of native ATCase tested with anti-serums specific against the enzyme or its isolated subunits indicate that the ligands provoke a major oonformational effect in the subunit to which they bind. This observation is more consistent with the “sequential ” model (Von Fellenberg et al., 1968). In addition, it has been reported by McClintock & Markus (1968, 1969) that conformational changes tested on the basis of trypsic digestibility and sulphydryl reactivity are consistent with the “sequential ” model in the presence of aspartate, and with the “concerted” model in the presence of succinate. Concerning more specifically the binding of the feedback inhibitor CTP, it has been well established (Changeux et al., 1968 ; Buckman, 1970 ; Winlund & Chamberlin, 1970; Winlund et al., 1971) that ATCase exhibits two kinds of sites for binding CTP, which differ by their affinity constants. It has not yet been shown unequivocally that these two distinct types of sites do not pre-exist on ATCase prior to the binding of the first molecule of CTP, but it is more likely that this difference in affinity for CTP reflects the existence of homotropic negative interactions. It has been shown that this type of interaction can be explained only in terms of the “sequential ” model (Conway & Koshland, 1968; Levitzki $ Koshland, 1969). In addition, it has been recently reported that at low concentration of CTP, ATCase shows also positive cooperative interactions between regulatory sites (Cook, 1972). The fact that in 2-ThioU-ATCase the homotropic and heterotropic effects are dissociated indicates that these two types of interactions must correspond at least in part to different molecular mechanisms. Several kinds of recently reported observations are leading to the same conclusion. It has been shown by Buckman (1970) that a unique spin label probe allows the detection of only one type of interaction, and that the detection of the other requires the use of a different probe. Hammes $ Wu (1971a,c) have extensively studied the binding to ATCase of sucoinate and BrCTP, an analog of the natural feedback inhibitor, by the temperature and concentration jumps technique. When these two ligands were together in the presence of the enzyme,

700

D. KERBIRIOU

AND

G. HERVti

the two relaxation processes corresponding to their bindings were observed. Such would not be the case if the two kinds of ligands would act by shifting in opposite directions the same conformational equilibrium. Furthermore, a conformational change of the metal-binding site seems to occur only during homotropic interactions between catalytic sites (J. Grifhn, personal communication). All these results show that the feedback inhibitor CTP is not acting by shifting a conformational equilibrium, which would be shifted in the opposite direction under aspartate influence during homotropic co-operative interactions and that the two types of interactions must correspond to distinct molecular mechanisms. However, some kind of relationship between them can play a role in the properties of native ATCase. A simple “two-state ” model cannot account for the behaviour of ATCase, but all the results can be explained in terms of a model based on the presence of four conformational states, as already suggested by Hammes t Wu (1971a,c). The two kinds of interaction can independently obey either a “concerted” or a “sequential ” mechanism. Since most of the results found in the literature tend to show that the homotropic interactions between catalytic sites meet the requirements of the “concerted” model, and that, on the contrary, the homotropic negative interactions between regulatory sites can be explained in terms of the “sequential ” model, the four

ATP?

11 11 CTP

ATP?

CTP

Pm. 6. “Four-state” model for ATCase. ATCase is represented under four states as already suggested by Hammes & Wu (197&c), but on the following assumptions: (i) T and R refer to conformations involved in homotropic oo-operative interactions between cetslytic sites, and hsving, respectively, low and high sffinity for aspartate (in terms of the “concerted” model of Monod et al., 1966); (ii) A and B refer to conformations involved in both homotropio negative i&erections between reguletory sites, and heterotropic antcrgonistio interactions between catalytic and regulatory sites. These two forms have, respectively, low and high ai%ity for espertate (in terms of the “sequentisl” model of Koshlend et al., 1966).

states can be represented as shown in Figure 6, where T and R refer to the conformations involved in the homotropic co-operative i&era&ions between catalytic sites, and A and B to the conformations involved in both the homotropic negative interactions between regulatory sites, and the heterotropic antagonistic interactions between catalytic and regulatory sites. In such a model, 2-ThioU-ATCase would be a pure RB form if, as is probable, the homotropio negative interactions between regulatory sites obey the “sequential” model, and would be an equilibrium between RB and RA forms in terms of the

NODIFIED

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TRAKSCARBAMYLASE

701

“concerted ” model. It is interesting to consider that ATP could act at the level of either the T + R or the A + B equilibrium, but it could also act independently by provoking another conformational change. The fact that 2-ThioU-ATCase can be stimulated by ATP suggests that the latter would be the case; however the same result would be obtained if the modified enzyme is an equilibrium between the RA and RB forms. It appears that ATCase is the object of a larger number of conformational changes than was first supposed, and that the activity of each catalytic site is the result of multiple influences. Some of these conformational changes occur in isolated catalytic or regulatory subunits (Collins & Stark, 1969; Eckfeldt et al., 1970; Hammes et aE., 1971; Kirschner t Schachman, 1971; Griffin et al., 1972), and the others require the complete quaternary structure of the enzyme. This complex set of conformational changes must provide a very sensitive and pliable process for regulation. The fact that a modification of the regulatory subunits is responsible for the behavior of 2-ThioU-ATCase shows that their quaternary structure is more important for the co-operative interactions between catalytic sites, than for the heterotropic interactions in which they are directly involved. This is confirmed by a recent study of the metal-binding site of the regulatory subunit by circular dichroism spectroscopy (J. Griffin, personal communication). The eventual effect of the 2-thiouracil-induced modification on the negative homotropic interactions between regulatory sites is now under investigation. The authors are indebted to Dr Gerhart (Berkeley) for providing the mutant used in these experiments, and to Drs Fromageot (Saclay), Stark (Stanford) and Griffin (Bethesda) for stimulating discussions. REFERENCES Buckman, T. (1970). &o&em&y, 9, 3255-3265. Changeux, J. P., Gerhart, J. C. & Schachman, H. K. (1968). Biochemistry, 7, 531-538. Changeux, J. P. & Rubin, M. M. (1968). Bioch,emistry, 7, 553-561. Cleland, W. W. (1967). A&u. in Enzymol. 29, l-32. Cohlberg, J. A., Pigiet, V. P., Jr & Schachman, H. K. (1972). Biochemistry, 11, 3396-3411. Collins, K. D. & Stark, G. R. (1969). J. Biol. Chem. 244, 1869-1877. Conway, A. & Koshland, D. E., Jr (1968). Biochemistry, 7, 4011-4023. Cook, R. A. (1972). Biochemtitry, 11, 3792-3797. Eckfeldt, J., Hammes, G. G., Mohr, S. C. & Wu, C. W. (1970). Biochemistry, 9, 3353-33~. Gerhart, J. C. (1970). In &went Topics in Cellular Regulation, vol. 2, pp. 275-325, Academic Press, New York and London. Gerhart, J. C. & Holoubek, H. (1967). J. Biol. Chem. 242, 288c-2892. Gerhart, J. C. & Pardee, A. B. (1962). J. Bid. Chem. 237, 891-896. Gerhart, J. C. & Pardee, A. B. (1964). Fed. Proc. Fed. Amer. Sot. Exp. Biol. 3, 727-735. Gerhart, J. C. & Schachman, H. K. (1965). Biochemistry, 4, 1054-1062. Gerhart, J. C. & Schachman, H. K. (1968). Biochemistry, 7, 538-552. Griffin, J. H., Rosenbusch, J. P., Weber, K. K. & Blout, E. R. (1972). J. Biol. Chem. 247, 6482-6490. Hammes, G. G., Porter, R. W. & Stark, G. R. (1971). Biochemistry, 10, 104fSlO5O. Hammes, G. G. & Wu, C. W. (1971a). Biochemktry, 10, 1051-1057. Hammes, G. G. & Wu, C. W. (19715). Biochemistry, 10, 2150-2156. Hammes, G. G. & Wu, C. W. (1971c). Scknce, 172, 12051211. Hill, A. V. (1913). B&hem. J. 7, 471-480. Jacobson, G. R. & Stark, G. R. (1972). In The Enzymes, 3rd edn., in the press, Academic Press, New York and London. Kerbirion. D. & Herve, G. (1972). J. Mol. Biol. 64, 379-392. 46

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Kirschner, K. (1971). In Current Topics in Cellular Regdudion, vol. 4, pp. 167-210, Academic Press, New York and London. Kirschner, M. W. & Schachman, H. K. (1971). 23iochemistry, 10, 1919-1926. Koshland, D. E., Jr, Nemethy, G. & Filmer, D. (1966). B&hem&dry, 5, 365-386. Levitzki, A. & Koshland, D. E., Jr (1969). Proc. Nut. Acad. Sci., U.S.A. 62, 1121-1128. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265-275. McClintock, D. K. & Markus, G. (1968). J. Biol. Chem. 243, 2855-2862. McClintock, D. K. & Markus, G. (1969). J. Bid. Chem. 244, 36-42. Monod, J., Wyman, J. & Changeux, J. P. (1966). J. Mol. Bid. 12, 88-118. Nelbach, M. E., Pigiet, V. P., Jr., Gerhart, J. C. & Schachman, H. K. (1972). Biochemistry, 11, 315-327. Perbal, B. & Herve, G. (1972). J. Mol. Bid. 70, 511-529. Porter, R. W., Modebe, M. 0. & Stark, G. R. (1969). J. Bid. Chsm. 244, 1846-1859. Rosenbusch, J. P. Q Weber, K. (1971a). J. Biol. Chem. 246, 1644-1657. Rosenbusch, J. P. & Weber, K. (1971b). Proc. Nat. Acad. Sk., U.S.A. 68, 1019-1023. Von Fellenberg, R., Bethell, M. R., Jones, M. E. & Levine, L. (1968). Biochemktry, 7, 4322-4329.

Weitzman, P. D. J. & Wilson, I. B. (1966). J. Biol. Chem. 241, 5481-5489. Winlund, C. C. & Chamberlin, M. J. (1970). B&hem. Biophys. Res. Commun. Winlund Gray, C. & Chamberlin, M. J. (1971). And. Biochem. 41, 83-104.

40, 43-49.