J. Mol. BioE. (1991) 220, 789-799

Heterotropic

Interactions in Escherichia coli Aspartate Transcarbamylase

Subunit Interfaces Involved in CTP Inhibition

and ATP Activation

Xu Guang Xi’, Frarqoise van Vliet2, Moncef M. Ladjimi’ Bbnedicte de Wannemaeker2, Christine de Staercke’, Nicholas Glansdo& Andre Pibrard3, Raymond Cunin2 and Guy Her& ‘Laboratoire d%nzymoEogie, C.N.R.S. 91198 Gif-sur- Yvette, France ’ Laboratorium voor Microbiologic Vrije Universiteit Brussei 1070 BrU88el8 Belgium 3Laboratoire

(Received

de Microbiologic, Univeraitt! Libre de Bruxelles 1070 Brussels, Belgium 1 November

1990; accepted 8 April

1991)

In Escher&a coli aspartate transcarbamylase, each regulatory chain is involved in two kinds of interfaces with the catalytic chains, one with the neighbour catalytic chain which belongs to the same half of the molecule (RI-Cl type of interaction), the other one with a catalytic chain belonging to the other half of the molecule (RI44 type of interaction). In the present work, site-directed mutagenesis was used to investigate the involvement of the C-terminal region of the regulatory chain in the process of feed-back inhibition by CTP. Removal of the two last C-terminal residues of the regulatory chains is sufficient to abolish entirely the sensitivity of the enzyme to CTP. Thus, it appears that the contact between this region and the 240s loop of the catalytic chain (Rl-C4 type of interaction) is essential for the transmission of the regulatory signal which results from CTP binding to the regulatory site. None of the modifications made in the Rl-C4 interface altered the sensitivity of the enzyme to the activator ATP, suggesting that the effect of this nucleotide rather involves the Rl-Cl type of interface. These results are in agreement with the previously proposed interpretation that CTP and ATP do not simply act in inverse ways on the same equilibrium. Keywords: Aspartate transcarbamylase; allostery; co-operativity; protein subunit interactions; protein engineering

1. Introduction

In terms of structure, ATCase is made up of two catalytic trimers (catalytic subunits) that are maintained in contact through their interaction with three regulatory dimers (regulatory subunits) on which are located the regulatory sites. The reaction catalyzed by ATCase operates through an ordered mechanism in which carbamylphosphate binds first, followed by aspartate, with release of the products in the order carbamylaspartate then phosphate (Porter et al., 1969; Collins & Stark, 1969; Issaly et aZ., 1982; Hsuanyu & Wedler, 1987). ATCase shows homotropic co-operative interactions between the catalytic sites for aspartate binding and possibly catalysis. These interactions are explained by a transition of the enzyme from a conformation that

Escherichia coli aspartate transcarbamylase (ATCase?; EC.2.1.3.2) catalyzes the first step of pyrimidine biosynthesis, that is the carbamylation of the amino group of aspartate by carbamylphosphate. This reaction is modulated in response to the intracellular level of pyrimidine nucleotides. ATCase is extensively studied as a model for cooperativity and allostery and its properties have been recently reviewed (Allewell, 1989; Herve, 1989; Kantrowitz & Lipscomb, 1999). t Abbreviations used: ATCase, aspartate transcarbamylm; PSE, primary and secondary effect; kb, lo3 base-pairs. 789 0022-2836/91/150789-11

$03.c!o/0

0 1991 Academic Press Limited

790

X. G. Xi et al.

has a low affinity for aspartate to a conformation that has a high affinity for this substrate (Howlett & Schachman, 1977; Moody et al., 1979; Krause et al., 1987). The crystallographic structure of these two extreme conformations is known to a resolution of 2.4 A (1 A=91 nm) (Honzatko et al., 1982; Ke et aZ., 1984, 1988; Krause et al., 1987). CTP, the end-product of the pathway, inhibits ATCase activity. This feedback inhibition is synergistically enhanced in the presence of UTP, which by itself has no effect (Wild et al., 1989). On the contrary, ATP activates ATCase. This antagonism is assumed to play a role in maintaining a balance between the intracellular pools of purine and pyrimidine nucleotides. ATP and CTP bind competitively to the regulatory sites which are located in the N-terminal domain of the regulatory chains (Honzatko & Lipscomb, 1982). Information obtained by enzyme kinetics (London t Schmidt, 1972), X-ray crystallography (Honzatko et al., 1982) and nuclear magnetic resonance studies (Banerjee et al., 1985) indicate that both nucleotides bind the regulatory sites in the anti-conformation. The first models that were proposed to account for the regulatory properties of ATCase postulated that the nucleotides were acting on the same transition as that involved in the homotropic co-operative interactions between the catalytic sites for aspartate binding (Changeux & Rubin, 1968; Howlett et al., 1977). However, over the years, numerous indications have accumulated suggesting that such is not the case (for reviews, see Hsuanyu 6 Wedler, 1988; Allewell, 1989; Her&, 1989). On the basis of these results it has been proposed that, in the presence of the substrate aspartate, the nucleotides act through the combination of a primary and a secondary effect (PSE-mechanism; Thiry & Herve, 1978; Taut et al., 1982). The primary effect consists of the following: when the regulatory nucleotide binds to the regulatory site, it alters the affinity for aspartate of the nearest catalytic site (positively in the case of ATP, negatively in the case of CTP), through a local conformational change that does not involve the gross TeR quaternary structure transition. In the presence of a given concentration of aspartate this change in affinity alters the proportion of the catalytic sites that contain aspartate (saturation function P) thus modifying the ratio of the T and R states of the enzyme (secondary effect, Thiry & Herve, 1978; Taut et al., 1982). This mechanism was verified through X-ray solution scattering experiments (Herve et al., 1985). Using equilibrium isotope exchange kinetics, Hsuanyu BE Wedler (1988) came to that same conclusion that ATP and CTP do not act directly on the TeR equilibrium. Interestingly, these authors showed, in addition, that the two effecters alter the rate constant for binding of aspartate to the catalytic sites. The crystallographic structures of the T and R-states ligated with either ATP or CTP have been solved to a resolution of 2.6 and 2.8 A, respectively (Stevens et al., 1990; Gouaux et al., 1999). The ATP

ligated ATCase is in the T-state. None of the hydrogen bonds or other polar interactions characteristic of this state are disrupted upon ATP binding to the unliganded enzyme, confirming that ATP does not promote the transition from T to R. Upon ATP binding, however, the distance between the two catalytic trimers increases by 0.5 A, a change which is negligible when compared to the 11 A increase of that distance which occurs during the T to R transition. This small movement can still be considered as part of the primary effect, which by definition, has no direct influence on the T/R proportion. The primary-secondary effect mechanism (Thiry & Her&, 1978; Taut et al., 1982; Herve et al., 1985) implies that CTP and ATP do not act simply in opposite directions on the same transition. In accordance with this prediction, a mutant form of ATCase, called pAR5-ATCase, was isolated, in which the effects of ATP and CTP are uncoupled (Cunin et al., 1985; Ladjimi et al., 1985). In this mutant the last eight amino acid residues at the C terminus of the regulatory chain are replaced by a new sequence of six amino acid residues: pAR5-regulatory Normal-regulatory

145 146 147 148 149 150 151 152 153 Phe Tyr Thr Lys Leu Ala Leu Phe Scr His Am Val Val Leu Ala Asn

This modified form of ATCase is normally sensitive to the activator ATP. However, this activation is limited to the extent of the “primary effect”, since this enzyme does not show homotropic cooperative interactions between the catalytic sites for aspartate binding (Taut et al., 1982). On the contrary, pAR5-ATCase is insensitive to CTP, in spite of the fact that this nucleotide normally binds to the regulatory sites (Ladjimi et al., 1985). The regulatory chain (see Rl in Fig. 1) has two contacts with the catalytic chains (Ke et al., 1988). One is the Rl-Cl interaction within the same half of the molecule, the other is the Rl-C4 contact between the C-terminal region of the regulatory chain and the 236 to 245 region of the catalytic chain that belongs to the other half of the molecule (Fig. 1). This last interaction involves amino acid residues that belong to the H3’ helix of the C-terminal region of the regulatory chains. Two features of the pAR5-ATCase modification might be responsible for the lack of sensitivity of this enzyme to CTP. One is the shortening of the regulatory chain by two amino acid residues, the other one is the substitution of six amino acid residues between positions 146 and 151. In the wild-type enzyme, this region contains the 147 to 150 H3’ helix (Ke et al., 1988). Prediction of the three-dimensional structure of this region based on energy minimization (Cherfils et al., 1987) indicated that helix H3’ can still be formed in pAR5-ATCase, a conclusion which is in accordance with the fact that the amino acid replacements are rather conservative, except for the change of asparagine 148 into a lysine. The same calculations suggested that in pAR5-ATCase the terminal carboxyl group of the regulatory chains can no longer form a predicted ionic bond with

Heterotropic Interactions

in Aspartate Transcarbamylase

(a)

(b) F?gure 1. ’The RI-Cl and Rl&C4 interfaces in ATCase and the residues modified in this study. These pi’ ct>UI’f?S were obt ained Wit ;h an Evans & Sutherland graphic system using the atomic co-ordinates generously P’ro lvidec 1 bq w Lipscl omb 1. (a) the regulatory chain RI is represented in green and the two catalytic chains Cl and C4 in .elloM : and blu t?, RSF scti vely; the 146 to 153 sequence of the regulatory chain, which comprises helix H3’. is shown in r-e!k The 240s between Rl. Cl and C4.riZla 11.5’2. rAsi 1153. 10010 of c 1 is shown in purple. (b) Close-up view of the region of inkrfaces 328 oj FRI and cArg250 of C4 are shown in light blue.

792

X. G. Xi et al.

arginine 250 of the catalytic chains (Cherfils et al., 1987; Xi et al., 1990). Instead, this carboxyl group would be in a position to establish an intrachain ionic bond with lysine 28 of the allosteric domain (Cherfils et al., 1987), an interaction which, in wildtype ATCase, does exist but only in the R state (Ke et al., 1988). This, contrary to the wild-type T state, pAR5-ATCase cannot form the Rl-C4 interaction between the C-terminal region of the regulatory chain and the 236 to 245 region of the catalytic chain (Fig. 1) that is described by crystallography (Ke et al., 1988). Here, site-directed mutagenesis was used in order to determine which structural feature of pAR5ATCase is responsible for its lack of sensitivity to CTP. The results obtained show that the Rl-C4 interface is essential for the transmission of the primary effect of CTP. In contrast, disruption of this interface has no influence on the primary effect of ATP, which therefore, must involve the Rl-Cl interface.

2. Materials and Methods (a) Chemicals Carbamylphosphate (lithium salt), L-aapartate, adenosine triphosphate (sodium salt) and cytidine triphosphate (sodium salt) were purchased from Sigma Chemical Co.; Tris(hydroxymethyl)aminomethane (Tris) was from Merck and L-[U-‘4C]aspartate (300 mCi/mmol) was from CEA-Saclay. (b) Bacterial strains, phages and plasmids E. coli strains 151OG4PYRF* (ApyrBI, pyrF bradytrophic, thi; 1510G4 from Perbal & Hervb (1972), further modified in Brussels), EDKl104 (ara, Apro-lac, &A, thi, 1985) and ApurB, pyrF+, rpsL; Nowlan & Kantrowitz, JM103 (Alac-pro, supE, thi, &A, endA, sbcB15, hsdR4/ F’traD36, proAB, lacP, ZAM15; Messing & Vieira, 1982) were used. Bacteriophages M13ATCwt and M13ATCpAR5 are derivatives of M13mp9 (Messing & Vieira, 1982) harbouring the inserts shown in Fig. 2. Plasmids pAR5 (Cunin et al., 1985) and pPBh104 (Roof et aE., 1982) are both pBR322 derivatives. pEVATC is a derivative of pUC18 (Yanisch-Perron et al., 1985) harbouring pyrBI on the same insert m ml3mp9pyrB1wt. (c) DNA manipulations Standard DNA manipulations were perfomed as described in Maniatis et al. (1982). Restriction enzymes were purchased from Boehringer Pharma and Pharmacia LKB, calf intestine alkaline phosphataae and bacteriophage T4 DNA ligaae from Boehringer Pharma. Nucleotide sequencing on either single-stranded or gradient purified double-stranded plasmid DNA was performed by the dideoxy chain termination method (Sanger et al., 1977) using the T7 DNA Polymerase Sequencing system from Pharmacia LKB. (d) Oligonucleotide synthesis and oligonucleotide-directed mutagenesis Oligonucleotides were synthesized with a Biosearch Cyclone DNA Synthesizer using reagents purchased from

PYfBl P M13ATCwt

Ml3ATCpAR5

Bs

B(alH

1

I

II

P

Bs

B(a)H

I

1

f-

> B(b)

S 1

Ikb+

Figure 2. Restriction map of the pyrBI inserts of the Ml3 phage derivatives used for mutagenesis. The position and polarity of the pyrB and pyrl genes, coding for the catalytic and regulatory chains, respectively, is indicated at the top. The pAR5 insert contains some 1 phage DNA (continuous line). Symbols of restriction sites are: P, PstI; Bs, BetEII; B, BglII, (B(a) and B(b) identify 2 different BglII sites used in the genetic constructions); H, Hpal; S, SaZI, BH, BamHI.

MilliGen/Biosearch and then purified by polyacrylamide gel electrophoresis. Mutagenesis followed the methodology developed by Kramer et al. (1984). The required M13mp9rev DNA was a gift from Mr P. Cuvelier from Boehringer Pharma, Brussels. (e) Construction

ad

expression of mutant ATCases

Mutagenesis was performed on a 2.7 kb PstI-SaZI DNA fragment from [email protected] containing the pyrBZ genes, cloned into bacteriophage M13mp9, or, in the case of the pAR5 ATCase genes, onto a l-88 kb Pst-BamHI fragment from pAR5 (Fig. 2). After nucleotide sequence determination to check the presence of the desired mutation as well aa the absence of any unwanted alterations, a DNA cassette harbouring the mutation was subcloned into a suitably deleted, linearized and purified expression vector derived from pUC18, to reconstitute the pyrBI genes. The presence of the mutation was ascertained by sequencing the double-stranded plasmid DNA. The constructions were transformed into a pyrBZ, pyrF bradytrophic strain, which allows physiological derepression of the pyrimidine genes under conditions of uracil starvation and, therefore, maximization of pyrBI expression. The pAR5 mutation is a substitution of the 8 amino acid residues at the C-terminal of the regulatory chain of ATCase, by 6 residues read from 1 phage DNA (Cunin et al., 1985). The DNA flanking pgrBI on the pyrl side is therefore different from that present on wild-type E. coli chromosome (Fig. 2); this explains why a different subcloning strategy was followed for mutants prepared in wild-type and in pAR5 genetic backgrounds. Table 1 (columns 4 and 5) reports the restriction sites used to extract the DNA cassette harbouring the mutation from Ml3 and to insert it in the expression vector, respectively. (f) Enzyme assay The ATCase activity was measured aa described (Perbal t HervB, 1972), in the presence of 50 mm-Tris.HCl buffer (pH 8), and 5 mm-carbamylphosphate. The enzyme concentration was determined using the method described by Lowry et al. (1951) using bovine serum albumin as a

Heterotropic Interactions

in Aspartate Transcarbamylase

793

Table 1 Mutants prepared for the study of the RI-C4 interaction stram \VT- ha

Mutation

Genetic background

Deletion of

wild-type pAI

Insertion of

Bglll-Hgll 1

RgZII(a)-HgII

I(b)

BglII(a)-BarnHI

BgzlI(a)--Hglll(h)

,501hp

rAla1.52 and rAsn153

pAR.i.rAsn28

Deletion generated in expression vect,or

ti30 bp

rAla 152 and rAsn 153

pAHii.Ala.Asn

DNA cassette with mutation

rLys28 - > Asn

pAR5

B.~tE11LBamH1 1125 bp

HstEIILN~~/ll(b)

rLys28 - > Asn

wild-type

&tEII-HpaI

LlstEII-HpaI

704 bp cArg250 - > Asn

wild-type

BstEII-HpaI 704 hp

These mutants were prepared as described in Materials and Methods, bp, base-pairs: (a) and (h) identify 2 different BgUI sites used in the genetic constructions.

standard and taking into account the 20:/, overestimate that is given by this method (Kerbiriou et al., 1977). The specific activity of the different enzyme species are expressed as pmols of carbamylaspartate formed per h per mg of protein. The influence of ATP and CTP on the rate of reaction was determined as described (Kerbiriou & Herve, 1972; Thiry & Herve, 1978). The percentage of stimulation by ATP is expressed as follows:

(1) where V, is the rate of reaction in the absence of ATP and V” the rate of reaction in its presence. The percentage of inhibition by CTP is expressed as follows: ,)

-(V,-1/,)x100 0 -

C’,

-

(2)

where V, is the rate of reaction in the absence of CTP and I’, the rate of reaction in its presence.

3. Results (a) Rationale for the preparation

of mutants

The different mutants prepared in the course of this study are presented in Table 1. The structure of PARS-AT(:ase differs from that of the wild-type enzyme in two respects. One is the shortening of the regulatory chain by two amino acid residues. The other is the replacement of a six amino acid sequence. The respective influences of these two alterations were investigated as follows. First, the two terminal amino acids present in the wild-type regulatory chain were added at the terminus of the pAR5regulatory chain (pARti.Ala.Asn). Second, these two amino acids were deleted in the wild-type regulatory chain (WT- 2aa). Besides, in order to test for the putative influence of the predicted intrachain interaction between the terminal carboxyl group of pAR5-regulatory chain and lysine 28 (Cherfils et aE., 1987), the latter was replaced by an asparagine (pAR5rAsn28). As a control, the same substitution was made in the wild-type enzyme

(WT.rAsnSS). Furthermore, in order to investigate the putative involvement on CTP effect of the predicted interaction between the carboxy terminal of the wild-type regulatory chains and arginine 250 of the catalytic chains (Cherfils et al., 1987) this asparagine residue was replaced by an (WT.cAsn250). (b) Injluence of CTP on the mod(fied forms

of ATCasp The inkuence of CTP on the catalytic activity of the different mutants prepared in the course of this investigation was determined. The results obtained are shown in Figure 3 and Table 2. It appears that’ the removal of the two C-terminal residues of the wild-type regulatory chain abolishes completely the effect of CTP. In contrast, the addition of these two amino acids at the C-terminus of the pAR$regulat,ory chain restores entirely the effect of CTP, demonstrating the importance of the C-terminal region of these chains in the mechanism of feed-back inhibition by this nucleotide. The replacement of

Table 2 tn$Yuence of the effecters ATP a,nd C’TP on the activity of wild-type and modijed forms of ATCase

WT ATCase pARS.Ala.Asn pARB.rAsn28 WT.rAsn28 WT.cAsn250 pAR5-AT(:asr WT - 2aa The influence of the effecters ATP (11 mM) and CTP (15 mnr) was determined as indicated in the legends b Figs 3 and 4. The (‘4 effects are expressed as indicated in Materials and Methods.

794

X. G. Xi et al.

EO60-

O0.01 L

0.1

I KTPI (rnM)

IO

,

80 -

Figure 4. Antagonistic effect of CTP on the activation by ATP of ATCase and its modified forms. Wild-type ATCase and mutant enzymes were incubated under the conditions described in Materials and Methods, but in the presence of 1 mM-aspartate, 5 m&r-ATP and increasing amounts of CTP. (a) ATCase (0.4 pg); (0) pAR5 (02 pg); (A) WT-2aa (@2 fig).

60 40 -

20 -

I 0.1 CCTPl(mw)

IO

Figure 3. Influence of the feed-back inhibitor CTP on the activity of ATCase and its modified forms. The activity of ATCase and of the mutants was determined as described in Materials and Methods in the presence of increasing concentrations of CTP. The aspartate concentration was 5 mM in the case of wild-type ATCase (02 pg), pARB.Ala.Asn (@2 p(g), pAR5.rAsn28 (@2 pg) and WTcAsn250 (02 pg), and 2 mnr in the case of pAR5 (@l pg) and WT-2aa (01 pg). The percent inhibition is calculated as described in Materials and Methods. (a) (0) WT; (m) WT.cAsn250; (A) pAR5. (b) (0) pAR5. Ala.Asn; (A) WT- 2aa. (c) (0) pAR5.rAsn28; (A) WT.rAsn28.

lysine 28 by an asparagine residue in the pAR5regulatory chains prevents the formation of the predicted ionic interaction between this residue and the carboxy terminus of these modified chains (Cherfils et al., 1987). This substitution is sufficient

to restore entirely the effect of CTP on the activity of pAR5-ATCase. The significance of this result will be discussed further. The same substitution in the wild-type enzyme does not influence its sensitivity to CTP. This is also the case when arginine 250 is replaced by an asparagine residue. (c) The lack of CTP effect is not due to a decreased afinity of the regulatory sites for this nucleotide In order to verify that the lack of sensitivity to CTP of pAR5-ATCase and WT - 2aa is not due to a decreased affinity of their regulatory sites for CTP, were performed as competition experiments described (Ladjimi et al., 1985). In the case of wildtype ATCase, the competitive binding of CTP and ATP can be evidenced in the following way: when the rate of reaction is measured in the presence of a subsaturating concentration of ATP, increasing amounts of CTP decrease the influence of this activator, then provoke progressive inhibition (Fig. 4). In the case of pAR5-ATCase and WT-2aa, under the same conditions, CTP entirely abolishes the activation by ATP. The concentrations necessary to

Heterotropic Interactions

795

in Aspartate Transcarbamylase

(d) Injluence of ATP on the modified forms of ATCase Figure 5 shows that all the modified forms of ATCase used in this study are normally sensitive to ATP. As expected, in the cases of pAR5ATCase and WT-2aa, the stimulation by this nucleotide is limited to lOO%, that is the extent of the primary effect of this nucleotide (Thiry & Herve, 1978; Taut et al., 1982; Herve, 1989). This maximal 100% stimulation is that exhibited by the wild-type enzyme in the presence of a concentration of aspartate or analogue sufficient to shift entirely the TeR equilibrium towards the R form (Taut et al., 1982). This is also the maximal stimulation shown by the modified forms of ATCase that are stabilized in the R state (Taut et al., 1982; Ladjimi et al., 1985). In the same manner, the maximal effect of ATP on the

- (bl

activity

of

WT.rAsn28

and

WT.cAsn250

lies

between those of the T and R states, in accordance with the fact that these modified forms of ATCase show a decreased co-operativity for aspartate binding (Xi et aZ., 1990). These results indicate that the features of the Rl-C4 type of interaction (Fig. 1) that were modified in this work are not involved in the mechanism of stimulation by ATP.

250

200

(c) E

4. Discussion (a) CTP and ATP effects appear to involve diflerent subunit interfaces

150

100

50

0 o-01

0-I

1 [ATPI

10

(rntd)

Figure 5. Effect of ATP on the activity of ATCase and its modified forms. The assays were performed as described in Materials and Methods in the presence and absence of increasing concentrations of ATP. The aspartate concentration was 1 mn in the case of wild-type ATCase (0.2 pg), pARSrAsn28 (@2 pg), pARLAla.Asn (0.2 pg), WT.cAsn250 (02 pg) and WT.rAsn28 (@2 pg), and @67 mM in the case of pAR5 (01 pg) and WT-2aa (01 pg). The percentage of stimulation is calculated as

described in Materials and Methods. (a) (e) WT; (A) PARS. (b) (0) pARB.Ala.Asn; WT-2aa. (c) (0) pARS.rAsn28;

(m) WT.cAsn250; (A) WT.rAsn28.

(A)

reach half the maximal value of these two effects are not significantly different. Thus, CTP binds to the regulatory sites of the two modified forms of the enzyme with the same affinity that it has for the regulatory sites of the wild-type enzyme.

The results reported above show that all the modifications of the C terminus of the regulatory chain (pAR&ATCase, WT-2aa) that disrupt the Rl-C4 interaction, abolish the effect of CTP on the enzyme activity. Addition of the two normally present amino acid residues at the C terminus of the pAR5-regulatory chains, a modification which gives back to the regulatory chains their full length, restores completely the sensitivity to CTP. Taken together these results indicate that the interaction between the C-terminal region of the regulatory chain (Rl) and the 240s loop of the catalytic chain (C4) is essential for the transmission of the CTP signal. In contrast, none of the modifications made in this region has an influence on the effect of ATP on the enzyme activity. Since the only other described interface between the regulatory chain and the catalytic chain is RlCl, these results strongly suggest that the primary effect of CTP involves the Rl-C4 interface whereas the primary effect of ATP involves the RlCl interface (Fig. 1). It is well established that the T to R transition involved in the homotropic co-operative interactions between the catalytic sites (Ke et al., 1988) is accompanied by the disruption of the Rl-C4 interaction. Therefore, an implication of the above conclusion is that the R state would be insensitive to the influence of CTP. Another implication is that the ATP signal would reach the catalytic site through the carbamylphosphate binding domain

X. G. Xi et al

796

and that the CTP signal would reach this site through the aspartate binding domain. Among others, the RlC4 interaction involves a salt link between lysine 143 of the regulatory chain and aspartate 236 of the catalytic chain (Gouaux & Lipscomb, 1990). The specific disruption of this salt link through replacement of aspartate 236 by an alanine residue leads not only to the abolition of CTP effect, but also to that of ATP (Newton & Kantrowitz, 1990). This result suggests that the effect of ATP might also involve this ionic bond, an interpretation which is consistent with the fact that this bond is predicted to be conserved in pAR5 ATCase (Cherfils et al., 1987). However, the disruption of this ionic bond might alter an interaction between lysine 143 or a neighbour amino acid residue of Rl with some amino acid side-chain of Cl that is very close (Fig. 1). In this region, there is close contact between RI, Cl and C4. This ternary feature might be involved as a whole in the transmission of the ATP signal. A comparison of the properties of pAR5-ATCase, WT - 2aa and pAR5rAsn28 shows that, in the wildionic interaction type enzyme, the predicted between the C-terminal carboxyl group of the regulatory chains and residue Arg250 of the catalytic chains (Cherfils et al., 1987), is not a requirement for the effect of CTP. This is shown by the fact that the replacement of Lys28 by an asparagine residue in pAR5-ATCase is sufficient to restore this effect in this modified form of the enzyme, in spite of the fact that the regulatory chains are still shorter by two amino acid residues, and thus, are most probably unable to establish an ionic bond with Arg250. In pAR5-ATCase and WT - 2aa, in agreement with the prediction of Cherfils et al. (1987) the interaction between the C terminus of the regulatory chains and Lys28 must keep the C-terminal region of the zinc domain in contact with the allosteric domain, thus preventing its interaction with region 236 to 245 of the catalytic chain, an interaction which appears to be essential for inhibition by CTP.

catalytic sites by aspartate or its analogues (Griffin et al., 1973; Foote & Schachman, 1985). It is induced in that sense that it seems to involve an “induced fit” promoted by the binding of aspartate to the catalytic sites, and that the homotropic co-operative interactions cannot be explained in terms of a simple two-state pre-existing equilibrium only. This is shown in particular by the fact that two pseudosubstrates of the enzyme, L-cysteine-sulfinate (Foote et al., 1985) and I,-alanosine (Raillon et al., 1985) are used by ATCase but are unable to promote the transition to the R state. Carbamylaspartate, one of the two substrates in the reverse reaction behaves in exactly the same way (Foote & Lipscomb, 1981). Thus, in the absence of a pre-existing equilibrium, a modification of the energy of interaction between the subunits could occur without having a significant effect on the quaternary structure. Tn such a case, the effecters might act solely through a local conformational change which would alter the interface between the regulatory and catalytic subunits, thus facilitating or hindering the gross quaternary structure change that would be provoked only as the result of substrate binding to the catalytic sites. In other words, in the case of ATP for instance, the primary effect would decrease the strength of interaction between the regulatory and catalytic chains. Consequently the complete shift to the R state would require a lower degree of occupation of the catalytic sites by aspartate. CTP would provoke the opposite effect. An interesting aspect of this putative mechanism which can be called “effector-modulated transition”, is that the local conformational change induced by the binding of the nucleotide to the regulatory site needs only to extend to the regulatory chain-catalytic chain interface. This feature would be consistent with the fact that no structural consequence of ATP or CTP binding to the regulatory sites could be detected at the level of the catalytic site, at the present resolution of crystallographic studies (Stevens et aE., 1990; Gouaux et al., 1990).

(b) Primary-secondary effects or ejfectormodulated transition The involvement of different subunit interfaces in the mechanism of action of ATP and CTP is fully consistent with the proposed mechanism of heterotropic interaction by primary-secondary effects (Thiry $ Herve, 1978; Taut et al., 1982; Herve et al., 1985), which implies that ATP and CTP do not act simply in reverse ways on the transition but rather by promoting different conformational changes. Another possible mechanism would be consistent with the conclusion that ATP and CTP do not directly influence the proportion of T and R states. This proposal is based on the fact that in ATCase the transition involved in the homotropic co-operative interactions between the catalytic sites appears to be not only concerted but induced. It is concerted in that sense that the full transition towards the R state is observed well before the saturation of the

(c) Do ATP and CTP have a direct effect on the T=R transition? The very smali effects that ATP has on the T quaternary state and that CTP has on the R quaternary state was recently discussed in view of the possible mechanisms for the heterotropic interactions (Stevens & Lipscomb, 1990). It was proposed that the effecters alter the substrate affinity by perturbing a quaternary state, although a putative influence of that small perturbation on the T/R proportion has still to be demonstrated (Stevens et aZ., 1990). These interesting results contribute to define the questions that must now be asked concerning the mechanisms of heterotropic interactions: does the 0.5 A variation of the distance between the catalytic trimers have a direct effect on the affinity of the catalytic sites for aspartate? Does

Heterotropic Interactions

in Aspartate Transcarbamylase

this @5 A change have a direct effect on the T/R proportion? It has been reported by Eisenstein et al. (1990) that in a mutant in which lysine 143 is replaced by an alanine residue, ATP and CTP have an effect on an observation which was the T/R proportion, taken by the authors as evidence that the same should occur in the wild-type enzyme. This work also shows that, in the absence of ligands, this enzyme has a quaternary structure that lies between those of the T and R states, an observation which is consistent with the fact that in this form of enzyme the mutation has eliminated the lysine 143-aspartate 236 ionic bond which is supposed to stabilize the T state. Also in the same work, it is shown that carbamylphosphate alone is able to entirely shift the quaternary structure to the R state, something that this substrate is unable to do in the wild-type enzyme (HervB et aE., 1985). There are several examples of this phenomenon; similar observations were made in the case of pAR5ATCase (Cherfils et al., 1987) and Glu239Gln-ATCase ((iouaux et al., 19X9; Taut et al., 1990). These observations indicate that in modified forms of ATCase in which the T state is destabilized, and in which. consequently, the energy barrier to the transition to R must be smaller than in the wildtype, some physiological or even non-physiological ligands are able to promote the quaternary structure transition, something that they are not able to do in the wild-type enzyme. In other words, the results reported by Eisenstein et al. (1990) show that indeed ATP and CTP have a direct effect on the quaternary structure of the Lys143-+Ala mutant, but certainly do not show that. such is the case in the wild-type enzyme. Any direct evidence of such a phenomenon is still lacking. Tn contrast, there are numerous indications that the nucleotides do not act directly on the gross quaternary structure change (Thlry $ Her& 1978; Taut et al., 1982; Her&, 1989; Hsuanyu & Wedler, 1988). The results reported here actually lead to the same conclusion. Tt was shown previously that pAR5ATCase and WT - 2aa do not exhibit detectable co-operativity and that, they behave like the R form of the enzyme (Xi et id.. 1990). If the modification of the C-terminal region of the regulatory chain had simply shifted the T+R equilibrium to the R form, and if the effecters were simply acting by altering this equilibrium, the consequences of the modification would be a decreased influence of ATP and an increased influence of CTP, a prediction which is in total contradiction to the behaviour of the modified enzymes.

Gif-sur-Yvette

for allowing

797 and helping to use the VAX

and PS390. References Allewell, N. M. (1989). Eacherichia coli aspartate structure. energetics, and transcarbamoylase: catalytic and regulatory mechanisms. Annu. Rev. Biophys. Biophys. Chem. 18, 71.-92. Baillon, J., Taut, P. & Her+ G. (1985). L-alanosine: for Eacherichia coli a noncooperative substrate aspartate transcarbamylase. Biochemistry. 24. 7182-7187. Banerjee, A., Levy, H. R., Levy. G. C. & Chan. W. W. C. (1985). Conformations of bound nucleoside triphosphate effecters in aspartate transcarbamylase. Evidence for the London-Schmidt model by transferred nuclear Overheuser effects. Biochemistry. 24, 1593-1598. Changeux, J. P. &, Rubin, M. M. (1968). Allosteric interactions in aspartate transcarbamylase. III. Interpretation of experimental data in t,erms of the model of Monod, Wyman. and Changeux. Biochemistry,

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Gouaux, J. E.. Stevens, R. C. Ke. H. M. 8: Lipscomb. This investigation was supported by the Centre National de la Recherche Scientifique, grants from the European Economic Community (BAP-0478-F; BAP-0345-B), a grant from NATO for travel expenses (0364/X8). a grant from the Belgian FKFO, and a fellowship from the Centre de Recherehe et d’Etude des Charbonnages de France to X.G.X. We thank M. A. Delsuc. B Amoux and P. Clapier from the ICSN at

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Edited by A. R. Fersht

Heterotropic interactions in Escherichia coli aspartate transcarbamylase. Subunit interfaces involved in CTP inhibition and ATP activation.

In Escherichia coli aspartate transcarbamylase, each regulatory chain is involved in two kinds of interfaces with the catalytic chains, one with the n...
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