. ~, Iltlllll+-;'l-

I

I 411

Effect of

I,Illih|l~,

J J Jl% IIU~4~

I'JllJ i ,,d,,I,Hliqi o f I tll.'l'¢,l|~ Ih ,~.h,.tlil~.,d ~,,,~ltl+..~ I ~ l I | ' ~ * , ) l 'it 11)t~%1% tw)l I'~l~'t I'tJtww,'~'~l '

I'+~)l

~,1 ~l,

-ketoglutaratc and

structurztl analogues on hysterctic

propcrttes of : -kctoglutarate dehydrogenase V I. llun~k, ().(i. R o m a s h arid V S Gon~a/ko~,,i

J he bur,~t el pl'odq~,l ,l~.~.UllltlJ,ttl~,~h th,r111~ lilt: I~.(iJ) rt.,i,.IJo,I ',,,,l'., IIl~,e'lhg,llCd It h,,ts N.,.II ~Jlo~,t n II,q to J~' Ihe o b h g , l t o r ' , fe.atur~, of ~. ~t.,I),~l~ hut m l ~ i,, ~,,hcn m,.r¢,~ml~ tile ¢11,t~, n1¢ "L,It|lr.llltolt I!~ K(~ %trtl~.ttlr,iI illhth'Ll~LIt'~ t'~f K ( # 411(I t h e " q l g r o t l p iltOdllli.AtlOll ~ u p p r e , ~ the IIiii1.11 |ltl(~l ',~,ltllollt I'~r¢~,~.lllltlF ~.,tt.tJ'~ .~1~, I lie ic,~tll:~i crier.lined ire i1'~ r t~,Otll t~l" t h e c'~,'~tcn,.¢ o r I h c rcgtd.ll~,r~ ~ t c fi,1 I'nt~thng I'~.( i d l | d iI,i q f t l t . l t | r iJ ,Ill th~ldtl¢~ C~S¢lltl,tl for II} ~te[etlc p r o l ~ r l , e ' ~ ~ f Ix( ,I )

", Keloylui,u.,te d¢ll~,dlogcn,l~¢ I l~'qcrcl~, propert) ", Ixetoglular d e in.llogt~¢ %11 group, I~,¢l~al,dor~ site

2

1 I N q - R O I ) U C ] I()N KC, D, ,1 ~.Ol~lponcnt el the ~11ult~en/~ntc K(.;I) ~.olllpie\, c,ltaly,,c,, the Ill st and rate-dote1 m11ung ~tep el K(; o\ld,~tl~c dc,.arbt~xylat~on IiIdlv~dual KGD ~.,ltdl~ses tile lea~.tton in tile plesence of ,ut~fi¢l,iI election 'acceptol ', Both ii1 the model sy,,tcm and ~ hell the KGD cornp i e \ function,, ,Is a ~ hole there ~', a dec~ c,l~e in K G D a~.ti~lty, caused nc~thc, by tile ~ub,,tZ,ltC depletion dtld the p r o d u c t ,tccumulatlon nor by oh,raging the ollgomcl ~c structure el K G D [i] T w o nldcpendcnt nle~.h,lnlm~s el O i l / y a l e tLl:lCttVatlOIl have been e~tabhshed corre,,pending to two stage,, o f the process [1] Tile slo~,, one I~ m o r e

cxp]essed

i11 t h e n l o d c l

syMelll, whole llrCvel-

,,lble ina~.tlVatlon due to he\acy,qllotcrl,ate i.', observed On the contrary, the ta~t stage mamtc~t~ ~(selt to the ,,ame degree both tla the model reaction and in the natural one; it may be reversed and plo;ceds only dullag catalysis Thl~ reactivation leads ~o the burst in the p l o d u c t a c c u m u l a t i o n during KG oxidative decarboxylatton, suggesting the kinetlcally slow ttansluon o f K G D during catalysis. Such enzymes are leferrcd to as hystereuc ones and the slow changes m then properties arc supposed to be i m p o r t a n t for regulanng the c o m ple× processes In r i v e [2]. In the present m~estlgatlon the hysteretlc properties ot K G D were shown to be reduced by increasing K G c o n c e n t r a t i o n and suppressed m the presence of structural analogues o f KG This regulation a p p e a r s to be reahzed through the binding o f K G or Its structural analogues to the site, d f f f ~ e n t f r o m the catalytic one. Correspondence addre~ V I Bun]k, Department of Btotherm~try, Mo~cow State Um~,ers~ty, Mosco~ 119899, USSR Abbreviations KGD, c~-ketoglutarate dehvdrogenase, KG, ~-ketoglatarate

Pabhshed by Elsevier Science Pubhshers 13 V (Biomedical Division)

MA 1 [ R I A I . S A N D M L F [ I O I ) S

[ h e ~.hcntl~.,ll', u,,¢tl 'Acre ol~hllllCd Iro111 Ihe folJo~,~,,lll-, ,,[~tlr~.c,, I~(J rrolll %¢1"~,,i, I'mt,l',"d!.lltl he~,,,lt.~dllofel f,tl¢ fi't}lll M c r , . k . the ,~tl-tl~,tl~i'dl ,-in,lie/due ~, o f tlic ~l.ll'l'~tf.lIc froltl %lglll,i l"he ~olllj",otlllds v, el,g' Of the fillC',t i~r,|tl¢ ,t~,|ll,lblc b.(,l) ~a,~ [~olalcd from plk.¢Oll br¢,l~t I11tl'.~J¢ ,t', III (~} 1%(;[),l~ll~,l I~, ~,l ~, d¢lCfltlin¢,l ~,~,llh I h c ,tfhft,.i i1 cle~.ti o n ,t,,,~.¢ptor h¢~,a~.~,allofcr

r,tt¢ (0 7 niM) ii1 () (I I~ %1 pot,|f,',ltlltl Ilho~,ph,tt¢ bLiffcr, pl-I 6 ~ Tile ,,,Ul",¢'~ el protltl~.l .t~.~.mlttti.~tlml '¢',c1¢ It'l,Ll~,/¢tl b) tile IlIOthfl,,.,'lllOll t)l' I lugg¢llh¢lt11", I11¢I h o d 141 A~,~.orthllg to It the~ ~ ere ¢11,,1tied ii1 n t i m e iili¢1% ,ll~ o f t 20 '~ ~,~,'llelt the tll tl.h~,ltlOII dill mg I%(,,D r¢,b.ll011 ~,,t', nol de~,.[ 1bell b) [Jig' kllletl~% Of" tile fir',t ord¢l i t2,11.tlOll, the Ill¢{}lod of R,| ~, ,tad I~o~llJdlld 151 '~*,,l~ tl'~cd IIit ~.onll'nlt,ltlOll,~ ~ e r ~ p e r f o r m e d

OI1 ,In IIIM ~.Ollll~,ttlbl¢ pcr,,oll,d ,,.Ollll'hllCr ~,,,th the 'M.ItlICAI)' pro ~ r , t m l It,J IIllthll I Lie o f prothh.I ,tkt, llllllll,IHOfl ',A 1%(.telCrllllllC(.[ [3} C~,. It ,IpOl,llll/b~ 1he ~,~l ~,e o b t , n n g d V.,ll h t11¢ ~.,ll~.tll,t|ed t.Ofl,q If'It ',. ,tltlc~ Ill

t i l e / e r e t1111~' ">H-group,, o f h O D ~ele illothfled 1~ ,t a.lnlll iit~.tlha 11.I.111 v, Ith a n e{ltlH11olar tlU,111111~ e l .l I~%dro'~,~,[llerCtll'lbelt/Ol~ ,[~.ld ill

0 1 N'I pot,t~tm'i pho,,ph,tle, pit 6 ()

3, R E S U L T S TIle semllogar~tlam~c plots of hexacyanoferrate reduction during K G D reaction with different K G concentratlons are shown in Fig I. According to the m e t h o d of H u g g e n h e l m (see section 2), changes m optical density at 420 n m during the fi×ed consecutwe time intervals ale plotted versus reaction time. It can be seen f r o m Fig. 1 that the quantity of the product synthesized m the s a m e time (t = 20 s) is decreased m the course of the ~eact~on (with increasing n). T w o stages o f the process are evident at high concentrations o f K G (F~g. 1, hnes 3-5), which is m accordance with earlier exp e r i m e n t s [I]. But decreasing the KG concentration leads to the disappearance of the fast stage o f m a c t w a lion in such a m a n n e r that at small substrate concentration pracucally only the slow stage is displayed (Fig 1, hne 1). This stage appears to be reduced by hexac y a n o f e r r a t e , as was shown earher [1].

147

~i.))|IIII~. 2"+'I, 111111|I'+I ~

4.0

lalmai,, l'J')l

I i W', l I I II l't',

~.,tl,ll),q,+ t,llC ,ind ,.oFl~C,luet'lll), lhc t,tt¢ ol the I t l l e t -

,., 0420.10 3

,,,OllS,¢l~.ItHl

I'~CIV.,¢¢I,

the

~ralM,',ll',,

CI11~,111¢ ",Ill-++",ll'+klq

~,Otnl'q¢',, ,uv, I the ftcc ¢tl/,~mc I', UlU,.ll }-"l'q,ltCl Ih,uI the t',lt¢ ~)1 lll~.' |',l~.l tthl,,.It~,,lltOll l'{+".,,..¢lltI~, ~,~,¢ h,l~,c s.ho','i, tl tx,%