Commiunication

Crystallilzation and Preliminary

X-Ray Diffraction Studies of an

Inactive Mutant AspartateTranscarbamoylase from

Escherichia coli*

tifo p ulu icatitln. F mici rx 11). 198 1)

Rosalind Kim, Ti-Sheng Young, H. K. Schachmant-,

and Sung-Hou Kim

-o m the of

LI boiit'otllrx. oral? the B o /ogy(tiertxof

A mutant aspartate transcarbamoylase, ATCaSe2M,has been crystallized at neutral pH. The mutant enzyme

has a single substitution of aspartic acid in place of

glycine within the catalytic chain and show's not only

the loss of enzyme activity but also marked changes in

the chemical reactivity of several amino acid residues

and weakening of interaction betw een regulatory and

catalytic subunits. Despite these differences, the mu-

tant enzyme crystals have the same space group and

cell parameters as the wild type crystals grown at pH

5.9.

Regulation oif the biosx ~nthesis of pxrmdnsin K.sclhctnou '11 15 achiexved in part H) the action of' aspartate

t i'aris,catI,)amiox las,e (c.ar-bam--ox lp1hosp)hate:L-asp.artate car-

h)amiox,lti-raHf'eras.e. EC 2.1.32')~xwhich c.ara,lx zes the formationoIf c,arbl-anm-x-1 aspartate as the fir-st comminitted step in the1pat hxx,ax' leading to pxr'1inudi(ines (11 2). ATCasel wxhich exhibitsa signl(idal dependente oIf enizx'me-i at tix tv on subs;trate con-

(-entiattono and is inhibited Itv Cu11 (as xxel a-, actixvatedi fixA'TP1). is composed of six catal>vtic and six regulatorx pl)ylxl)plid(e chains xx,,hit-h ate organized xx.ithini the ititact enzxyme, astxx-(o catalx til'- trimiiel s a~nd thr-ee regulator> diniiers (for rexviexwsee Refs. 3 XThN ax dliffraction scde to a r.esolutitn oIfabout 3Ahaxve xvtelded traces of the l)olxypeptidle chains xwithinthe intact enzy mei-i an(l the location of the six zinic lions boundto the regulatorx chalins (6).

Since research on niuttationallx altered prioteins, l)oidexsclxvaluable iii'inforationi relating structure ant i'function in allo-steric prIotelns(7 81. xxe haxve initiatedi X rax diffrat'tionstu-dies tin miutant formiis of ATCase. One mui-tant, ATCase--from E. t'd)/l strain HSS13,kxx ith the xf2.3 I allele is-, of plaritictilai' initerest since it lacks enzxvme actixvitx and th-e regulatorxchains appeal to lbe identical wxitli th-ose of the xxilci txypeenzxyme 1.9). Tlhe m-iutanit enzxyme has a single substitution ofasplartic acid in p)lace (Ifglxcine at potsitioIn 1215 'In the tentatixve

'Ibis investigation xasucpporitted ix- Puhlte Hc-alth Serx ict Re-clarch Giants Gjll121t9 NSI11744. and ('CA274-4 anId fix Nationlal

Sc-ic-nec Foundation iest-a c h Gtrant 1T'T ) lhe co-T of,ptubltcatiollIof, th-is atrtic It xwetcd-f xt inlldp lixb the paxmni(tiot(Ipagt- chat gt- ThiH artitt I intiHt ih'tf( be httcfxmat ketid 1i

tisc;nat n ac-cotdnct- xxit 18I'L SC. Set,ton 1 34 '-ll o itid(iitot

I'h- ahU- ttiO-c.td ate - ae satic incriiiIac.23It as suhscript a muitaniit t-nzx,tnt- of. aspartate ir-atis-

'I HP 141 0444444 .A.141 HI-MiIITxo- 11 of Mai pl) 44 4 -

u41~i,/u1i ., A

aioaid sequtence of the cataix,tic. chains kindly proxvitledby WV. Konigsherg, Department of Molecular Biophysics andBiochenmistry, Yale Universitv (10¾. This substitution, xxicappears to be at a region rem-ote fromi the residlues in theactixve site, not onIx causes a loss in actixvitx of the C,- suibuniitbuit also cauises m--arked chaniges in the reactixvitx of sexvei'alothei aminiio acid residues located at diverse r-egions of' thep)rotein 10. In alddition the interaction betxween the C iandcregulatorx subu-nits in ATCase is significantly xweaker than in

the v,xild tyl)e enixmzie (9).

EXPERIMENTAL PROCEDU)tRE S

A Va~tse,- was pLiritiled (troini E. co/i' strain HSa 1: ca xtgthe/141 1-B2 31 alle-le a'' ording to the prtorediuie of Wall ci I. 00 1.TheiiuifttlIi/\ oexx - i'al> zeie agains 40 x potass.iuii phIiphale11

hofTci at pH 7.0 conitaininiig 2 ma 2nmeicap)toethaiiol) and 0.2 mamEDTA~. wrsal'ainxas petrfollniedl o0ii solution (If Al(a i12 nig/ ml in the Hamie bof'f'er clnanngIa4x x~p)olx ctl-x- let,1c gix lALV 000 in a iIel1)ressl(~io pIlilt etqtilihrated ito 10'4xlxmi t

ylent- gixcoIl 1)x- the x ap)or dif Minet hod (11 1) The ix1as4II\-Utt -iz omctimes as large as mm11 <icoswI ithin 2 to xxweek Inl

a co111rooalIti41 4 -C. si-tlXtesalc-d inl g1ass cap)illniiitthatxticleaned aint piresilakedi in th-e samle hlisoIlntioIn aH thle xtli111oii buffci, andc the dlata coIllectiOln W1(1 also tlonie in 4C

A.s, shoxxn in- Fig. 1, the c-txstals haxve a rhombic shap11e xwitiha sule in the long dlimnsei.-,on about I mmn. St illphtog istaken wxith a r-otatinig aniode N ra> gener-ator- sbowx a difrc i0opattern to 2.5 A resolution. A precession photograph takenalong the 34fold axis is s;hoxx n,i in Fig. 2. The space group) andlcell parameters (obtained from- the precessionphtlgahaind the diffractonmeter ar.e as, f(ollowxxs: saegroup is 132 andlcell 1paramnieters atre a h 1 30.8 A; c =1981) A;: /90' .~ 121)~on a hexagonal settin-g.

'lhe space gr-oup) for ATCasei is 1identica,,l and the celll)aram-eters foi' the mu-i-tanit enz.'x mear xer nsmiar to thostlbtainedl for- the xwild txype enzix e crxystallized at pH oh(6).I1)ata to aA resolution haxve benobtained and the cleto(Ifh-ilgher. resolution dlata is~ill progress.

i)IS(CUSSiON'[he dleterm-ination of the structure of ATC'(ase,. of1spe-itil

signif'icance ixiexof' the striking changes tinte c iisligftrom a single substitution of'ani aspartli acid i'esidiie(

C

ON~~~~~~2"'V

Fit; I. Photomicrograph of cryvstals of an ATCase mutant,A'FCaSe21t. (itllhx-Iomi hApt 1hase at loxxrcg-itigh is 200)u loIng.

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4 lutant Aspartate Transcabarnox'lase Crvstals

Fit. 2. A 12 precession photograph taken along the 3-foldaxis of the rhombic erystal using Cu-Kui radiation.

fur. glycine in the catalytic polypeptide chains. This singlealteration at a position reemote from the residues at the actitesite results niot onlx in a loss of ( atalsvtic actixitv t also leadsto marked changes in the reactix it'- of residues at both theactixe site in the w ili tx-pe enzi-me andt at a )osition aconsitlerable distance from the actie site (10). Thus it appearsthat there are significant differences at the le-exl of the tertiarxs--tructures of the miiutanit anIcl wxilcl type proteins despite ecrv-tallizationi of both in the sanme space group. The change in thel)rimarx .structulre of the (ratalvtic 'hainrs is manifested also bx-a large weakening of the bonding domainis between the cata-lytic and regulator- r hains (9) ancl a .sutbstantial strengthening

of the intersubunit interactions xwithin the catalx ticsubuinits.It will be especiallx interesting to examine the structure of theregulatory chains in AlI'Case-,;, in relation to the x-il typeenzyme in viexw of the imiarked quaternarx- restraint exhibitedby the hxbrict enzm-ie molecules containing one xild tvpecatalytie subu-nit and one C,3 subunit along with three wildtx,pe regulatorx subtunits (12). On addition of the hisubstrateanalog, N (phosphvonacetxl) -L-asp)artate. crystals of the xwildty)e break apart but those of the miutant remiiain intact, agaiinmanifesting the chemiceal difference despite the fact that bothhave the same erystal space group. The high resolution crxstalstructures of the wild txpe and the mu-titant enzxymes xxill becompared in collaboration with Dr. Lipsconmis laboratorv atHarvard Unixversitv.

Ad fnous leclgn?ernts -We are indebted to Cel1ine Ho. for- thle prepa-rationi of t.he purified muilitanit enz". miie aspaartate tiane thaniox We231

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7799. Glerhart. J. C. aticl Pardee. A. B. (19621 J. Biol. (hn. 237. 891_

896.3. (Gerhart. J. C c197)(0tr Top ('Lr Regul. 2, )7-4. Jac(_)hl n G. 1a.nand Star . C. R. 1 9 ii in Thl e Ern xrnrf RnB() v e r.

P. 1) edi) 3rdl Ecl. Part B, Vol. IX. pl). 225-.308. Ac.ademie lPressN-ew York

.a. cha{ hal^lll H.c.H 1974 Hart cx I eeT 68, f-6 1136. Mon)iac o. 1-1. L. C'ra,-ford.*,J. L.. and Lipsecl N 19 )s Pur i

.'all/. Actad. Si iU.S .A. 75, a276 528))7. Sihulmiiani. R G_ Hophield. J. J.. antid ()Ogaxa,a. 19;) R()

Bzuphlx s 8, 325-420)8. Perotz. l. F. 1976i Br. Med. Baill. 32, 195- 2)89. Wall. K. A.. Fiatgaard, J. E. GibbonsL.. anti Schabhrmian 1-1. K.

(1979 J. Biol. (Chem. 254, 1 1910-1 119111). WalL K. A. anti Stha(hnai. 11. K. (199) -]. Biol. Cheat. 254.,

11917 1192611. Kim. S.-H-. ani Quigley G. J. 199) Mdthods En ool 59, S-2112. (Gabbons, L. Flaiiganiri. J. E.. arIcd Shl4 hi<jn H K.K. 7.n Iton

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R Kim, T S Young, H K Schachman and S H Kimaspartate transcarbamoylase from Escherichia coli.

Crystallization and preliminary X-ray diffraction studies of an inactive mutant

1981, 256:4691-4692.J. Biol. Chem. 

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