Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli

,I. Mol. Bid. (1985) 186, 715-724

Structure-Function Relationship in Allosteric Aspartate Carbamoyltransferase from Escherichia coli II. Involvement of the C-Terminal Region of the Regulatory Chain in Homotropic and Heterotropic Interactions Moncef M. Ladjimi’??, Char-s Ghellis2, Andre Feller3, Raymond Cunin4 Nicolas GlansdorfT4y , Andre Pi&rard3v4,5 and Guy Her& ’ Laboratoire 2 Laborcrtoire

d’En.zymologie,

de Physico-Chimie 3 Microbiologic,

4 Erfelqkheidsleer

C.N.R.S.,

des Protbines, Universiti

en Microbiologic,

5 Research Institute

91190 Gif-surUniversitt?

Yvette, Fra,nce

Paris XI,

Orsay. Frnncr

Libre de Bruxelles,

Belgium

F’rQe Cniversiteit,

B,russels, Belgium

of the CERIA-COOVI, 1 Avenue l?miEe Gryson B1070, Brussels, Belgium (Received 5 March

1985)

The modified aspartate transcarbamylase (ATCase) encoded by the transducing phage described by Cunin et al. has been purified to homogeneity. In this altered form of enzyme (pAR5-ATCase) the last eight amino acids of the C-terminal end of the regulatory chains are replaced by a sequence of six amino acids coded for by the ,? DNA. This modification has very informative consequences on the allosteric properties of ATCase. pAR5-ATCase lacks the homotropic co-operative interactions between the catalyt’ic sites for aspartate binding and is “frozen” in the R state. In addition. this altered form of enzyme is insensitive to the physiological feedback inhibitor CTP. in spite of the fact that this nucleotide binds normally to the regulatory sites. Conversely, pAR5-ATCase is fully sensitive to the activator ATP. However, this activation is limited to the extent of the previously described “primary effect” as expected from an ATCase form “frozen” in the R state. These results emphasize the importance of the three-dimensional structure of the and het’erot,ropic (‘-terminal region of the regulatory chains for both homotropic interactions. In addition, they indicate that the primary effects of CTP and ATP involve different features of the regulatory chain-catalytic chain interaction area.

1. Introduction

(1985). Concerning the regulatory properties of ATCaseS, it seems that the homotropic co-operative interactions between the catalytic sites for substrate binding can be explained by some kind of concerted transition from a low affinity conformation (T) to a high-affinity conformation (R) (Gerhart & Schachman, 1968; Hammes & Wu, 1971; Griffin et al., 1973; Kirschner & Schachman, 1973; Gibbons et al.. 1976; Howlett et al.. 1977). These two extreme conformations can be stabilized t’hrough experimental modification of proteinsolvent interactions (Dreyfus et al.. 1984). However,

Aspartate transcarbamylase catalyses the first reaction of the pyrimidine pathway, i.e. the carbamylation of the amino group of aspartate by carbamylphosphat,e. In Escherichia coli, this reaction is feedback inhibited by the end-product CTP and is stimulated by ATP. The structure and the properties of this allosteric enzyme have been extensively reviewed (Gerhart, 1970; Jacobson & Stark, 1973; Kantrowitz et aE., 1980a,b) and their relevant features are summarized by Cunin et al. t Present address: Department of Chemistry, College. Chestnut Hill, MA 02167, U.S.A. $ Abbreviations used: ATCase, aspartete transcarbamylase; caps, cyclohexylaminopropane sulfonic acid.

there are several indications that this transition does not correspond to a simple two-state thermodynamic equilibrium, which would be shifted toward the R

Boston

conformation

through

an exclusive

binding

of the

substrate to this form. Among them is the fact that t,he conformational change associated with the 715

002%2836/85/240715-10

$03.00/O

0

1985 Academic

Press Inc. (London)

Ltd.

homotropic c+o-operative int,cbractions between t’hc caatalytic: &es is characterized by an important shift in thts pH dependence of this reaction, showing that. a,t low aspartate concentration, it is the T caonformation that’ participates predominantly in thr catalysis (Gerhart & Pardee. 1964; Thiry B Her-G. 197X). This is also shown by the fact that in the reverse reaction, carhamylaspartate is appart,nt’iy unable to promote the allosteric transition. alt,hough it) has a higher afinity for the It conformation (Foote $ 1,ipscomb. 1981 ), Thrh sarnc’ kind of observation was made using rit,her I,-cysteine-sulfinate (Foote et al., 1985) or Lalanosine (Baillon et a,Z., 1985). aspartat*e analogurx in the forward reaction. As far as the hctrrot,ropic interactions art’ c~onccrned, it appears t>hat the effec%ors ATT’ and (‘TI’ do not act dire&l?- on t,he TG R transit,ion involved in the homotropic co-operative interacbtions for subst,rat,e binding. Instead thesr nuc~lrotid(ls act through a “primary &ect”. which is a locsal conformational change altering “site by site” the affinity of the catalytic sit)es for aspartate. and a “srcaondary rffect ” on the T + R transition, n-hich is mediated hy the substrate aspartate (Thiry & H~rvi~. 1978: Taut: rt ol., 1982). This mechanism was rrc*rntly (*onfirmed by S-ray scattering experiments (Herv6 it al.. 1985). As expected t)he secondary &&t does not occur in t,he absence of substrate OI in modified ATCases “frozen” in the R c*onfortnation (Kerbiriou & Her+. 1972. 1973: KcLrbiriou et al., 1977: (‘hat1 & Enns. 1981; Taut it al.. 1982). ‘l’h~ present knowledge of the amino acid st~cpt’n”fY of both the catalytic and regulatoq chains of ATCase (Weber, 1968: Konigsberg & Henderson. 1983: Hoover et rrl.. 1983; Schachtnan Pt cl/.. 1984: (lunin et trl.. 1984) and the detailed irifortrratiori csoncerning obtained t,llr thre(Jdimensional structurr of this enzyme (Honzatko r>t (11.. 1979, 1988: Honzatko & Lipscomb, 1982: Ke et al.. 1984) should now permit’ abtempt’s to correlate the above-mentioned regulatory features with structural changes. It w-as reported previousI> (Feller rt nl.. 1981) that a lnrql pyrH1 transducing phage directs the biosynthesis of a modified form of ATCase when infecting an E. coli strain deleted for t,he wild-type ATCase operon. (lunin d (11. (1985) have shown that the modificat,ion results from the fact that’ the sequence of nucleot’ides coding for t ht. last eight amino acids of the regulatory chain of -ATCase is replaced by a phage DNA sequence coding for six amino acids. The present report descaribrs the (~omplrt~~ purification of this modified form of ATCase and the extensive study of its csatalytica and altered regula,tory properties. The amino acid sequence at the (‘-t)rrminal end of the regulatory (*hain is in full agreement with the corresponding I>SLA srquenct~. The result>s obt’ainrd emphasize the importan(*e of thr (“-terminal region of the regulatjory cahain for t hr existence of both homotropic and heterotropica int,erac+ions in ATCasc.

Mutant ATCase Regulatory Chain.

Figure 1. Chromatographic purification step on DEAESephadex ASO. Cell extract obtained from t,he heat step (150 ml at) 68 mg prot,einjml) was put on a DEAE(60 cm x 3 cm) previousI) Srphadex A.70 c,olumn rqrtilibrat~ed with IO mwimidazol HCI buffer (pH 7) cwtttaining 0. I tnwp-mercaptoethanol and 0.35 M-KU. Thr elution was performed using the same buffer and IO ml frac*tiotts MWY collected. The ATCase activitv was tncwsutwf using I ~1 samples from each fraction d”ilut,etl 100 x (‘nits of twzytnt~ actirity are pmol carbamylaspartatr fortnrti,‘h. under the conditions used.

modified rata]>-tic subunits and 0.75 mg of nortnal or tnotlitietl zinvregulatory subunits were incubated in a total volume of 0.5 ml in the presence of Zx lW3 Mp~tttercal)toethattol and lW4 M-ZillC acetat)e for 15 min at 37 c’. The reconstituted enzymes were analysetl It> rlec+rophoresis on polyarrylatnidr gels. as describrd in section (e). and t)ested for their enzymatic properties.

Samples (3 trig) of pAR-regulatory subunits WP’IP incubated at Z’(” in thr presence of c.arbo~~Ete’Ettic~ase;\ and R (Ftrotei,ilenz?m~~ = *SO.for each carboxypeptidase) in 10 mwpotassium phosphatr buffer (pH i) containing I tn~l-B-mrrcar)torthanol and 0.1 m*t-EDT=\. in a total volume of I ml. From time to time. #-PI samples were taken in which thr rraction was stopped hy the addition of 10 ~1 of 0.5 M-H(‘1. After centrifugation of these saniJ)lrs. the supernatants were rvalwratrd to dryness. I)eproteittizatiotr of these satnples was achievrd by thv arid according to thta addition of IO”,, sulfosalicylic method of Alondino rt (11. (IS’il). Amino acid analyses were then performed using a Kont,ron amino acitl a post-column derivatization b) atral~vser. with ot,tttol)htllaladeh~(if~ as reagent.

3. Results (a) I’/tr(fimtion of pil RSA T(‘asr and dissociation iflto its mtnlytic rind rrgu~latory subuGt.s

pAR.!%ATCase strain KM I<[,

was prepared from t,he extracts of 1510(X, transformed by pAR5

II

Figure 2. I’ol~acr~lamide gel elf,c~t,rol)llorrsis of mutant and wit&type A’lY’ase and their subunits. Electrophoresis was performed as described in Materials and Methods. The samplrs aw: I. pAR5ATCase (IO pg): 2, AT(“asr (10 pa): 3. c*atalytic subunits (20 pg): 4. p;\R;i-cat,alytic subunits (I:’ L(g): 5. regulatory subunits (Ii pg): 6. pAR5regulatory subunits (I!) j.q).

plasmid. KM 131, 151OG1 is a thermoresistant derivative of strain KMHI, 1510 (F-jyrl1, Mu et&%) that produces no det~cctable amount of either 13. Perhal. subunit (G. Her\-6 Nr ATC’ase unpublished observation). A high production of pAR5ATC’a~se was ensured by the introduct ion of a acid dwarboxylase gwr) leak) /qrF (erotic mutation. The purification of the modifietl enzyme was obtained using a modification of the procedure described by Gerhart & Holoubek (1967). The heat.step was performed at 55°C AS in the c*ase of _“~thiourac,it-AT(‘ase (Kerbiriou & Hervb. 1972). Previous work by Feller et nl. (1981 ) showed that these cell extracts contain about equal proportions of native pAR.%ATC’ase and vatalyt’ic subunits. The on DEAEstep chromatographic purification Sephadex allows t)he virtually complete sef)aration of these two species (Fig. 1). The analysis of the two fractions by gel elect rophoresis showed that peak (I corresponds to native A4T(‘asr and peak 0 to trwes of subunits. The remainit~p catalytic catalytic subunits that might still wntatninate the pAR5ATC’ase fraction (peak (I) at this step are eliminated during the subsequent previ~)itation of the enzyme at its isoelectric point. Such a purified pAR5-AT(‘ase is stable and tlors not spontaneously dissociate filrttwr into its subunits. For experimental purpose the dissociation so-called p.4R~,?-c*atalyt ic of t.his enzyme into and pAR.5regulatory subunits n-as subunit’s performed according t,o the method of (:erhartj KHoloubek (1967). Figure 2 shows the result’ of the electrophoretic analysis of these different prepaw pAR5ca,talytic subunits and catalytic tions.

718

et al.

M. &I. Ladjimi

I/ (pmol 0.0

0.6

2.4

hei)

l-6 ,

0.8

I

(

I

A-d

1

2.4 ,

3.2 ,

0.06

0.24

2.5

0.2

0.02

0 Aspartate

concentrotton

1/ (pmoi

(mM)

h-‘I

W

(b)

(a i

Figure 3. Absence of homotropic co-operative interactions between the catalytic sites in pAR5-=\T(‘ase. pAR5-L\T(‘asts (0.06 pg) (0) and wild-type ATCase (0.24 pg) (A) were incubated under the standard conditions. but in the presence of increasing amounts of [14C]aspartate (from 0.66 to 26.6 IrIM). (a) Initial velocity (z*) as a function of asparta& concentration S (insert: corresponding double reciprocal plot); (b) Eadie plot.

migrat’e to t,he same position. The migration of the pARSregulatory subunit,s is slightIT slower t)han that of the wild-type regulator! subumts, suggesting a discrete difference of charge bet,ween these two proteins. This interpretation will be confirmed in sectlion (f). However, no difference bet,ween normal and pAR5-subunits migration could be detected when they were analysed by subunits

electrophoresis

under denat,uring

(b) pAR5-ATCase co-operative

conditions.

does not exhibit homotropic interactions between the cntalytic xites

Figure 3(a) shows t)hat the aspartate saturation curve of pAR5ATCase is hyperbolic, indicating the lack of homotropic co-operative interactions between t,he catalytic sites for t,he binding of the substrate.

This

result

is confirmed

by the

caorre-

sponding double reciprocal plot (insert) and Eadie plot (Fig. 3(b)). The calculated value of t,he K, for aspartate is 12.7 2 2.1 rnM based on five determina,tions. a value that, is significantly lower than the value found for both catalytic subunits and PARScatalytic subunit’s (20 mM), indicating that PARSATCase is frozen in t’he R conformation. (c) 7’he pff-dependence of pAR5-ATCase activity characteristic of the R conformation of ‘4TCasr

is

It has been previously established that the T and R extreme conformations of ATCase differ in their pH dependence for catalysis (Gerhart & Pardee. 1964: Kerbiriou & Herv6. 1972: Thiry & Her&. 1978). The optimum

pH for cat~alytic

in t’hr presence of low concentrations

activity

is 6.8

of aspartate

(T conformation) c*oncentrations

and X.2 in the prest~nce of high of aspartate

(R conformation).

The

isolated catalytic subunit,s and modified AT(‘ases that arp frozen it1 the R conformation rxhibit atI the aspartate optimum pH of 8.2 wha,tever caoncent,rat,ion (Kerbiriou & Hervt;. 1972. 1973: Kerbiriou of al., 1977: Taut. 1982)t. Figure 1- shows that. even in the presence of 5m~aspartat~e. pAR5ATCase has a rnaximal activity at pH 8.2. in cont)rast to normal ATCase. This result wnfirms that pAR,5-AT(‘ase is frozen in t.he R’ conformation. (cl) pA 125-A 7Y’nsti is normally nctivatwr A TP

se~nsitiw to the

As mentioned in the Introduction, the influence of ATP on the catalytic activity of ATCase is explained through a local conformational change (primary effect) that is amplified through the influence of the substrate, aspartate (secondary effect) (Thiry & Her&, 1978: Taut rt a,l.. 1982; HervC et a‘., 1985). Modified enzymes t,hat are frozen in t)he R conformation exhibit only the primary effect of this nucleotide (Taut et ul.. 1982: Taut. 1982). Consequently the maximal stimulation of ATCase by ATP is 2500/b in the presence of lov, concentrations of aspartate (T conformation) and only 1000/b in the presence of high concentrations of aspartate (R8 conformation): modified ATCases frozen in the R conformation are maximally stimulated by 1009;. regardless of the nspartatr t Tt is \vort,h noting t)hat the rxact position of thtasc, two maxima depends slightly on the nature of the buff~l, system which is used.

i

719

Mutant ATCase Regulatory Chain. II

7

6

9

8

IO

PH

Figure 4. pH dependence of pAREi-ATCase activity. pAR,5-ATCase (0.17 pg) and wild-type ATCase (0.15 pg) were incaubatrd and their activity determined under the conditions described in Materials and Methods, but in the presence of 5 mM-[ 14C]asparbate and 50 mw-Tris/bisTris/Caps buffer at, the pH value indicated: (A) wild-type ATCase: (0) pAR5-ATCase.

concentration (Taut et al., 1982). The influence of increasing concentrations of ATP on ATCase and

pAR5-ATCase aspartate

activities

is shown

in the presence of 1 mM-

in Figure

5. As expected,

the

maximal stimulation of ATCase reaches 250°,b, a value that is characteristic of the T conformation.

Figure 6. Effect of CTP on pAR5-ATCase activity. Wild-type pAR5-ATCase (0.06 pg) were conditions described in Materials presence of I mM-aspartate and CTP: (A) wild-type ATCase: (a)

wild-type AT(‘ase and ATcase (0.19 pg) and incubated under the and Methods. hut in the increasing amounts of pAR5-ATCase.

In contrast pAR5-ATCase shows the maximal stimulation of lOO%, which is characteristic of the R conformation. This result indicates that PARSATCase is normally sensitive to the stimulatory influence of ATP.

(e) pAR&ATCase is insensitive feedback inhibitor CTP 250

50

0 0.01

0.1

I [ATPI

Figure 5. Effect of ATP on pARSAT(‘asr activity. Wild-type pAR5-ATCasr (0.06 ,LJ~) were conditions drsrribed in Materials presence of I m.n-aspartate and ATP: (A) wild-type ATCase: (0)

IO (IVIM)

wild-type ATCase and ATCase (0.19 pg) and incubated under the and Methods, but in the increasing amounts of pARB-BTCase.

to the

It can be seen in Figure 6 that, contrary to what is observed in the case of ATCase, pAR5-ATCase activity is not reduced by increasing concentrations of the feedback inhibitor CTP. Such a lack of inhibition could result either from the absence of (‘TP binding to the regulatory sites of the modified enzyme or from an ineffective binding. In order t’o distinguish between these two possibilities, competition experiments were performed. Tt has been previously established that the CTP and ATP binding sites overlap (Honzatko et al., 1979: Honzatko & Lipscomb, 1982) and that these two nucleotides bind consequently competitively to the regulat#ory sites (Thiry & Hervk, 1978). The activity of ATCase and pAR5 ATCase was measured in the absence and presence of 1 mM-ATP, which provokes the stimulation of these enzymes by 1107; and 55:;. respect,ively. and increasing amounts of CTP were added t,o a series of such samples. Figure 7 shows that, in the case of ATCase? not only does the addition of (ITP abolish the stimulatory influence of ATP but the highest concentrations provoke the expected inhibition. (:onversely: in the case of pAR5-AT(‘ase, CTP addition abolishes the influenc>e of ATP but does

[CTP]

(mM)

Figure 7. EfCc~t of’(‘TJ’ on tbr stimulation of wild-tyJw AT( ‘BW and J’AR5-i\T(‘astL activity by ATJ’. IViJd-type .IT(‘aw (0.1.5 pg) and J,;\J<.5 AT(‘ase (0.06 &) \vrr’r in(wbattvl undrr the cwnditions dewribt~d ill Matwials and M&hods. but in t,hr presrnw of 1 mzl-aspartattx. J n1w ATP and increasing arnount,s of (‘TP: (A) wild-tyJw ATC’ase; (0) pAR,5-ATC’asr.

not provokv any inhibition t’vtw at the highrst cwnwntrations used. The dependent of’ thrsr effects on (‘TP concentrat,ion indicates that (‘TT’ hinds to the regulatory sites of p.AR15-AT(‘asr with an affinity that must he wry ~IOSP or equal to that for the regulat~ory sites of’ ATCase, Thus. it appears that (‘TI’ hinds to thv regulator!sites of‘ @KSATCaw. hut this binding dew not lravr ate!’ influence on the affinity of thfa wtalytic sites for, aspart.atc~.

In or&r to determine whit-Jl of’ the two types of’ subunits is responsible for the alt~ered proprt~irs of’ and normal .A’J’(‘asc JAR:?-AT( ‘aw, this enzvmr were dissociated int)o th;ir c>a,talytic+ and regulatory subunit.s and hybrid rwonst it,utracl enzymes wt>rt’ prepared as describwl in Materials and Methods. Figure X shows the result of thr eJwtroJJhorrticb

Mutant ATCase Regulatory Chain.

0

IO Aspartote

II

721

20 concentrptmn

(mM 1

Figure 9. Aspartatr saturation curves of t’he hybrid enzymes made up from normal and pAR5-catalytic2 and regulatory subunits. Hybrid XTCase molecules made up from normal and pAR85-catalytic and regulatory suhunits were prepared. mti their aspart’ate saturation curves were determined using 0.15 /.q of pAR5-regulat~ory/normalcatalytic

(0)

eatal@

(A).

and 0.15 pg normalLregulatory/pAR51.. initial

velocity.

I

0.1 0

I

I1

IO

1 20 Ttme

et nl.. 1982). Since the expression of the pAR;iATC’ase operon is repressed ire viva by the addition of nracil t’o the growth medium in E. ~oli cells infected with inrgl pyrBI, the alteration result’ing from the transduction process was most probabl?

0.2

0 6

7

8

9

IO

PH

Figure 10. pHiactivity profile of the hybrid rnzymes made up from normal and pARScatalytic and regulatory subunits. The pH dependence for activity of the two hybrid ATCase species described in Fig. 9 was determined as indic*ated in Materials and Methods using 5 mw aspartat.e and 0.15 pg enzyme. (0) PA=regulatory/normal-catalytic. normal-regulatory/ (A) pAR5-catalytic,

I’, initial velocity.

I

I

30

I 40

I

I 50

(h)

Figure 11. Kinetics of sequential amino acid release from the (’ terminus of pAR,R-regulatory suhunits under the influence of carbox~prptidases. The rate of amino ari(l wlease from the (’ terminus of pAR5-regulatory subunits under t,he influence of ~arhox~pe~)titlases A and B were determined as described in Materials and Methods. (a) Release as a function of time: (b) logarithmic plot, of Ieucine and alanine release (a,verage of three drterminations): (A) leucine: (0) alanine: (V) Iysine: (m) threonine: (0) t,yrosine ( x ) phenylalanine.

concerning t)he distal end of t’his operon that corresponds to the (‘-terminal region of the regulatory chain. These observations led us to analgse the amino acid sequence of this region of the protein. The rates of release of the amino acids from the C-terminal region of the pARSregulator> subunits by carboxgpept’idases A a,nd K were det’ermined as described in Materials and Methods and compared with amino acid release from normal regulatory subunits. Figure 11(a) presents the kinetics of the appearance of free amino acids. from \vhich their sequence has been deduced. The ambiguity resulting from the presence of two Irucine residues is solved by the corresponding logarithmic plot (Ambler. 1972). which provides the following ratrl constants (Fig. I l(b)): KLeul : 0.7 moI h - ’ 1 KLeuz: 0411 mol h-‘. K,,,:

042 mol h - ’

722

lll.

sequent Thus, the C-terminal regulatory subunits appears t,o be:

pARSregulatory. Sormal-reguiatory.

of

N.

Ladjimi

pAR,Y

et al.

c’arbosvr)rf)tidas:es t,han the wrresponding in normal AT(‘aw.

prpt.i(lr

145 I46 147 148 149 150 151 151 153 . .Phe-T?:r-Thr-Lyvs-Leu-AlaLeu .Phr-Ser- His- Asn-Val- Val- lieu-Ala- Asrl

These results are in full agreement

with

4. Discussion

the DNA

sequence determination reported by Cunin et 01. (1985). These kinetic dat)a further show that the substituted peptide is more accessible to the

Takct~ tog&her. the rrsults reported hrrcb a11d in (‘unin rf the paper b\ al ( I985) st1ow regulat.orJ. that Olf? alttwd unambiguously Equaiorial

domain

Allosteric effector

Figure 12. Scahrmatic thrrc-dimensional strucature of the int~rwtiny regions of tlw c.atalytic :tr~tl rcyttl;rtc,r,y c.l~uirr~ (from Honzatko ~1al.. 198%.with permission from LT. Lipscomb). (a) Swondary struvturr of a catalJ.tic, c,t~;lirl-rrprllator,~ chain unit:

(b) cw-carbons trace

in the region

of contxct

betwwn

catalytic>

and regulator~~

chains.

Mutant ATCase Regulatory Chain. II result from a properties of pAR5ATCase modification of the C-terminal region of the regulatory chain. This is the first description of a mutant bearing a modification of the regulatory chain, all the previous attempts to select such mutants having been unsuccessful (O’Donovan b. Gerhart. 1972, 1973: Syvanen & Roth, 1973). First of all this modified enzyme provides an additional example of the separation of homotropic and heterotropic int)eractions in ATCase, a phenomenon that has been obtained previously as the result of chemical modifications (Kerbiriou & Her&. 1972. 1973; Kantrowitz et al., 1977; Kerbiriou et al., 1977: Kantrowitz 8r Lipscomb, 1977; Enns & Chan, 1978, 1979; Chan & Enns, 1979). Moreover, pAR5ATCase clearly exhibits uncoupling of ATP and CYTP effects. On the basis that the modified ATCases that are frozen in the R state show only what has been previously described as the primary effect of these nucleotides (Thiry & Hervk, 1978; Taut et al., 1982; He& et nl., 1985), it can be concluded that. in terms of that primary effect, pAR5-ATCase responds normally to the binding of the activator ATP. In contrast. the binding of the feedback inhibitor CTP does not influence the affinity of the catalytic site for aspartate. The primary effect of the nucleotides is a local conformational change distinct from that) involved in the homotropic cooperative interactions between the catalytic sites (Thiry bz He&, 1978: Taut et al., 1982; Hervk et al.. 1985). This effect resulting from the binding of XTP or (‘TP t,o the regulatory sites is exerted at the nearest catalytic site, which is about 60 A away. Interestingly, in pAR&ATCase, this signal operates in the case of ATP but cannot be transmitted in the case of CTP, as a consequence of the modification of the C-terminal region of t’he regulatory chain. This observation indicates that the transmission of the signals promoted by the binding of either ATP or C’TP involves different features of the contact area bet,ween the catalytic and regulatory chains. This conclusion is in accordance with the previously reported indications that ATP and CTP do not act in inverse ways on the same conformational change (\+‘ong, 1971: Winlund-Gray et al.. 1973; Heyde et (11.. 1973a.b: Wedler & Gasser, 1974; Chan & Enns, 1981; Kantrowitz et al., 1981; Burz B Allewel, 1982: Silver et al.. 1983: Shanley et al., 1984). The extensive X-ray crystallographic study of ATCase (Honzatko et al., 1982; Ke et al., 1984) has revealed some side-chain interactions that are impaired in pAR5-ATCase, resulting in the altered regulatory properties of the enzyme. This is the case for the cc-helix structure of the C-terminal end of the regulatory chain, from histidine 147 to alanine 152, including a polar interaction bet’ween histidine 147 and asparaginr 148 (Fig. 12). In wild-type ATCase, the interactions between the two domains of the rrgulat,ory chains are hydrophobic; leucine 32 and phenglalanine 33 (helix Hl’) and leucine 76 and tryosine 77 (helix HZ’) from the allosteric effector domain, pacbk against valine 106, leucine 107 (strand S6’) and leueinr 161 (helix H3’) from t’he

723

zinc domain (Fig. 12). These interactions must ensure a relative rigidity within the regulatory chain. This rigidity might be necessary t,o impose the constraints t’hat decrease the affinity of the catalytic sites for aspartate in the T state. It is interesting to consider that the lack of helix H3’ in pAR5-ATCase might allow more flexibility of t)he pAR5-regulatory chain, leading to a weaker interaction between the regulatory and catalytic subunits. This phenomenon would give the catalytic sites t’heir maximal affinit’y for aspartate, so abolishing the homotropic co-operative interact,ions for the binding of this substrate. Leucine 151 is present in pAR5-ATCase, as in the wild-type enzyme (Fig. 12). However, its orientation is probably inadequate, since it’ is located at the C of the modified hexapeptide. This terminus difference in orientation is suggested by the fact’ that t’he C-terminal sequence of pAR5-regulatory chain is degraded by carboxppeptidase much more rapidly that that, of t’he normal regulatory chain. Some amino acid side-chains that are not direct)ly implicat’ed in t,he modification but are in the vicinity of the modified peptide could consequently adopt an abnormal orientation. These alterat’ions might also be responsible for t’he lack of homotropic co-operative interactions and the insensitivit’y to CTP. This is the case for arginine 128 and glutamic acid 144 (Fig. 12), which are interacting t,hrough a polar link. This is also the case for lysine 139. tyrosine 140, glutamic acid 14% and phenylalanine 145. which are involved in the interactions between the catraraman for reading and improving the manuscript.

References ,\mbler. 1%. P. (197”). Bfethods Enzymol. 25, 143-154. Raillon. J.. Taue, P. & HervB, G. (1985). Biochemistry, in t,he press. Beck. (‘. F. & Hewlett. (i. ,J. (1977). ./. Mol. f?iol. 111.

I-li. &II-Z, I). S. bz Allewel, ?u‘. M. (198%). Bioc~hrmistry. 21. .t Bti47-6655. (‘han. 12’. &‘. (‘. & Enns. (:. A. (1979). (‘a?rnd. J. Wiochem. 57, iWG305.

(‘ban, 1V. \V. (1. & Enns, (:. A. (1981). (‘and. 59. 4(il-%8.

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