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Activation of isocitrate lyase and triosephosphate dehydrogenase in Azotobacter vinelandii extracts
22~
BIOCHIMICA ET BIOPHYSICA ACTA
BBA XZI6 9 ACTIVATION OF ISOCITRATE LYASE DEHYDROGENASE IN AZOTOBACTER
AND TRIOSEPHOSPItATE VINELANDII EXTRACTS
]. R. I,:HI'~NEDY AND M. J. DII.WORTH" Instffute t~f /Igricult,m, University of $VeJtern A u~fralia, Ned,lands {,4 ustralia)
(Received June t9th, t962} SUMMARY [s0citrate ]yase is 20treed in Azolobact, er v i n e l a n d i i during g r o w t h on acetate. Reported requirements of isocitrate ]yase for Mg 2+ a n d cysteine have been confirmed, bu~ E D T A could replace cysteine for activation, a n d did so more efficiently. This o b x ' i a t ~ the serious interference by cysteipe in glyoxylic acid m e a s u r e m e n t . Isocitrate lyase has a h a l f - m a x i m u m velocity at & 6 - z o -6 M threo-I,s-isocitrate a n d is not inhibited b y high DL-isocitrate c o n c e n t r a t i o n (up to 8 raM). Triosephosphate dehydrogenase has been identified in a m m o n i u m s u l p h a t e fi'actions of crude e x t r a c t s of Azotobacter. This e n z y m e is also a c t i v a t e d b y E D T A nlore efficiently t h a n b y cysteine. It is concluded t h a t the~e activations b y cysteine a n d E D T A are the result of binding of inhibitor}' h e a v y metals in the as,say m e d i u m , T h e possible n a t u r e of ."filch metals is discussed. INTRODUCTION l)uring .~tUdics of oxygen t o x i c i t y on e n z y m e s concerned in o x i d a t i v e p a t h w a y s in Azotobacter v i n e l a n d i i , suitable conditions for assay of i-socitrate lyase (Ls-i.~)citrate g l y o x y l a t e lya.~e EC 4- z.3. I) a n d trio~ephosphate d e h y d r o g e n a s e (v-glycera]dehyde 3-phospoate : NAD oxidoreducta~e, EC x , z . ~ . z z ) were sought. lsocitrate lya.~e~ cataly.~e~ the reversible cleavage of isocitric acid to glyoxylic acict and .~,~cc:.ai= ;,,rid. and has been d e m o n s t r a t e d previously in ext,'acts ~f acetategrown A. vinMa,:dtt a n d A . agi/eL Keqtiircmen¢.~ for a d i v a l e n t m e t a i ion a n d thiol c o m p o u n d s for m a x i m a l a c t i v i t y h a v e been reDortedt-4; SMITH AND GUNSALUSt f o u n d t h a t g l u t a t h i o n e was slightly less active t h a n equimolar cysteine in m e e t i n g the l a t t e r requirement. A similar thiol requirement for a c t i v a t i e n of triosephc~phate dehydrogena_se from mammaUa.n systems has been reported6, 4, t h o u g h SEGAL AND I$OVV:R ~ showed t h a t muscle e n z y m e could be a c t i v a t e d successfully b y E D T A in place of cysteine.
" /~rcscnt addre~ : .Microbiology Unit. D e t r i m e n t of Biochem~try, Univer.~ity of Oxford, Oxford ((;ruat B r / t a / n ) . Bi~im.
Bio~kF~. Ac~. 67 ( t ~ 3 ) Z26-z39
I%OCI1-RATE
I.YA~E
AND
TRI()SF.PIIOSPIlA'rE
DEHYDROGENASE
227
B e c a u s e t h i o l c t ) m p o u n d s w o u l d c o m p l i c a t e st,:dies w i t h g a s e o u s o x y g e n , t h e s e finding:, h a v e b e e n e x a m i n e d fiw A z , ) t o b a c c e r e n z y m e s . S e r i o u s d i s a d v a n t a g e s in t h e use o f c y s t e i n e for i s ( ~ i t r a t e ly:t~e a c t i v a t i c m h a v e .been d e m o n s t r a t e d . METHODS
Bacteriological F o r p r e p a r . ' , t i o n ~,f (..:~tri'.t'.t.~ ~:t>rlt,tt,~,tlg i~,~citrate l y a s e , a s t r a i n or" Azotobacter vinelamlii d e s i g n a t e d S w;m u.~tt ~. A. ~,inelandii 0 w a s u . ~ d for p r e p a r m i o n of t r i o ~ e p h o s p h a t e d e h y d r o g e n a t e . C u l t u r e s w e r e prc.-~erved b y v a e t t u m - d r y i n g 9 a n d c a r r i e d o n M6 a g a r m e d i u m 1° for h r o t h in(~;ulati(m. L a r g e r q u a n t i t i e s o f cells for p r e p a r a t i o n o f cell-free e x t r a c t s w e r e g r o w n on a c e t a t e m e d i u m , o f t h e f o l l o w i n g compf~siti(m, lx-r 1: N a a c e t a t e , 20 g ; K H z P O 4, o . t 5 g; N a , , H l ' ( ) s r x 2 Ha(), ~.x 5 g; M g S O , - 7 H2(), o.2 g ; C a S O a - z H , O , 0.05 g; Na.M,u-~ . ,~ ,,03 g; E I ~ T A tron c(mqflex, o.()x 5 g; Na(21. o . r g; C u $ O 4 - 5 H 2 0 , 0. 4 r a g ; M n S O , - 4 H~O. z.o m S ; Z n S O s . 7 1[,O. o.8 m ~ : [[.~BO~, 2. 4 m g ; w i t h t h e e x t r a c t f r o m a u t o c l a v i n g [2. 5 g o f p e e l e d potat,~ in z 5 g of w a t e r f o r 4 ° r a i n a t 5 Lbfinz. S t e r i l i s a t i o n w a s b y h e a t . w i t h t h e p h o s p h a t e s ~terilised s e p a r a t e l y t o p r e v e n t p r e c i p i t a t i o n . I n o uia ~ v e l e 5'~/0 v o l u m e s o f c u l t u r e g r o w n on n i t r o g e n - d e f i c i e n t s u c r o s e m e d i u m (.X122) ( ~ e ref. 8). W i t h a c e t a t e as c a r b o n , o u r c e , a l k a l i w a s p r o d u c e d a n d t h e p H w a s c h e c k e d p e r i o d i c a l l y d u r i n g g r o w t h a n d ret u r n e d t o n e u t r a l b y t h e a d d i t i o n o f ~terile HCI. C u l t u r e ~ w e r e g r o w n in 2o-1 P y r e x j a r s u n d e r fi)r(:e[t a e r a t i o n at c o n t r o l l e d t e m p e r a t u r e . Cells w e r e h a r v e s t e d a f t e r 36 h g r o w t h b y c e n t r i f u g a t i o n w i t h a Ser,;all m(~deI S S - r . w i t h c o n t i n u o u s - f l o w a t t a c h m e n t , a n d .~tored a t - - 2 0 ", Extract_~ c o n t a i n i n g triosepho.c, p h a t e dehy(lrt)gena.se w e r e o b t a i n e d f r o m cells g r o w n o n M22.
Preparation of cell-free extracts Cell-free e x t r a c t s w e r e o b t a i n e d b y a l u m i n a g r i n d i n g o f frozen cells u. Tlne g r o u n d cell m a t e r i a l w a s ~ u s p c n d e d irl o . o r 5 M p h o s p h a t e b u f f e r ( p H 7.0) at a r:lte c o r r e s p o n d i n g to x g w e t w e i g h t o f cells pet 20 m i o f b u f f e r , a n d c e n t r i f u g e d first for xo r a i n a t 4000 × g to r e m o v e a l u m i n a particle, s a n d cell ~tebris, a n d t h e n for x0 rain a t 2o o(~) × g. T h e s u p e r n a t a n t wiL~ u~ed [br e n z y m e a ~ s a y a n d c o n t a i n e d a p p r o x . 3 m g o f p r o t e i n / m l . T h e s e o p e r a t i o n ~ were c a r r i e d o u t a t or b e l o w 3 :~.
Reagents D i s t i l l e d w a t e r u_sed t h r o u g h o u t w a s deioni.~xt , n B i ~ - D e m i n r o l i t resin ( P e r m u t i t Co.) t o a specific c o n d u c t a n c e o f o . x / ~ m h o / c m . T h e a l u m i n a u s e d fl~r p r e p a r a t i o n of cell-free e x t r a c t s w a s A l o x i t e - 6 o o ( T h e C a r b o r u n d u m Co.. M a n c h e s t e r ) , w h i c h h a d b e e n w a s h e d w i t h o.ox M E D T A ( p H 8.o). r i n s e d s e v e r a l tim~'~ w i t h d e i o n i s e d w a t e r . a n d d r i e d a t 8o ¢. B i o c h e m i c a l s w e r e o b t a i n e d f r o m tile fi;Lh)wing .~ources: c r y s t a a l i n e l y s o z y m e a n d o x a l o a c e t i c a c i d , f r o m N u t r i t i o n a l Biochem::-~ds C o r p o r a t i o n ; N A D a n d N A D P f r o m S i g m a C h e m i c a l Co. ; Naa-threo-osr-.s-i.~Jcitrate f r o m F l u k a C h e m i s c h e F a b r i k ; g l y o x y l i e a c i d m o n o h y d r a t e , f r o m L i g h t a n d Co. ; a n d g l y c e r a l d e h y d e r - b r o m i d e 3 - p h o s p h a t e d i o x a n e o x o n i u m s a l t , f r o m C a l i f o r n i a F o u n d a t i o n for B i o c h e m i c a l Researt:h.
Bi~him. BJophy+..-Jan, 67 ([963) 2=6-230
22~;
I. R
KENNEDY,
M. J . DI1.WORTEi
Aualvtical Isocitrate l y a ~ a c t i v i t y was d e t e r m i n e d b y m e a s u r e m e n t o f gl yoxyl i c acid f o r m a t i o n by the m e t h o d o f MCF^DDEN A.~r~ HOWFS aS. w hi ch involves c01orimelry of the x,5-diphenyl-formaz~an carbx, xylic acid f o r m e d on o x i d a t i o n t~f g l y o x y l i c acid l~ht,nylhydrazone with Kol:e{CN)~. T o e ns ure r e p r o d u c i b i l i t y o f results, t i m e of h e a t i ng was carefully .~tandardised at 5 rain. A reference sam pl e of g l y o x y l i c acid m o n o h v d r a t e wa_s 99-5 O¢ 7o pur e b y t i t r i m e t r i c assay xa. DL-Isocitric acid was t)t;°~n pure, as s ho w n by N A D P r e d u c t i o n w i t h a c r u d e l>reparation o f isocitric d e h y d r o g e n a s e f r o m a c e t a t e - g r o w n A . vinelandii. N A D P r e d u c t i o n wa.~ followed at 34 ° m/x in a B e c k m a n - D U s p e c t r o p h o t o m e t e r , with cell c o m p a r t m e n t at .30°. T h e p r o c e d u r e follc~wed t h a t of I{ORNI3ERGla. with o.o6 M Tris buffer (p14 7.5) replacing p h o s p h a t e . In th e initial e x p e r i m e n t s , t h e a s s a y s y s t e m for isocitrate lyase was similar t o t h a t described b y SstrrH AND GI;N.~AIA.:St, c a r r i e d o u t in T h u n b e r g tubes, or in flasks a t t a c h e d to the m a n o m e t e r s o f a VVarburg r e s p i r o m e t e r . T h e final v o l u m e was 3.o ml, ~sith nitrogen as t h e gas p h a s e t h r u u g h o u t . R e a c t i o n was s t a r t e d b y a d d i t i o n of isocitrate f r o m t he side a r m a f t e r t e m p e r a t u r e e q u i l i b r a t i o n at 30°, a n d s t o p p e d with o.2 ml o f 80o/o (w,'v) t r i c hl or oacet i c acid. A ft er p r o t e i n wag r e m o v e d b y eent r i f u g a t i o n , an aliquot of the s u p e r n a t a n t was a n a l y s e d for g l y o x y l i c acid. Controls to which .~ubstrate was adde~l a f t e r t r i c h l o r o a c e t l c acid a d d i t i o n c o n t a i n e d negligible amounts of glyoxyiate. T r i o s e p h o s p h a t e dehydrogena_~e was ;Lssayed b y o b s e r v a t i o n o f N A D r e d u c tion at 34 ° m/~. T h e a.~say s y s t e m follows t h e p r o c e d u r e o f KREESa, which uses c v s t ein e as a c t i v a t o r . T h e p u r i t y of c o m m e r c i a l DL-glyceraidehyde 3-pho.~pbate i -bromide d i o x a n e o x o n i u m .,~alt was d e t e r m i n e d with A z o t o h a c t e r e n z y m e ; it a.~sayed at b7%. "l'rioscphosphate d e h y d r o g e n a s e was p r e p a r e d from 6 g wet weight of sucrosegl o w n cells, which were g r o u n d for eo min with t5 g o f alumina. T h e g r o u n d m a t e r i a l was take~ up in 48 rnl of 0.03 M p y r o p h o s p h a t c buffer (pH 8.7), c o n t a i n i n g 2.5 ml of o.o~ .',1 n i c o t i n a m i d e , .,,,,--' centrifugtxi for x2 rain at 7ooo x g. E n z y m e was p a r t ially purified from t h e s u p e r n a t a n t b y a m m o n i u m s u l p h a t e f r a c t i o n a t i a n , mc~t a c t i v i t y being p r e c i p i t a t e d b e t w e e n 0.6 and 0,8 ~ t u r a t i o n , T h e e n z y m e was stable f~)r at leagt 3 month.~ at zo °. F u r t h e r purification was n o t u n d e r t a k e n , ~iPce a c t i v i t y could be satisfactt~rily d e t e r m i n e d w i t h o u t o b s e r v a b l e r e o x i d a t i o n of NAt)H.. Pro, to'in wag m*,ngllrod b~' th~ methexl o f I.OWRV el al. x¢. with crystallit~e l v s o z v m e as s t a n d a r d . ('tw-.matagraphy K e t o acids were c o n v e r t e d into their 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e s• a n d s e p a r a t e d b y ;Lscending c h r o m a t o g r a p h y on W h a t m a n No. 4 paper. S o l v e n t s were b u t a n o l e t h a n o l - w a t e r (7 o : x o : 2 o ) Is a n d i s o p r o p a n o l - a m m o n i u m h y d r o x i d e - w a t e r (xoo: 5: Io) 16. F o r co]our d e v e l o p m e n t of '-rots, dried p a p e r s were d i p p e d in z°./o N a O H in 900,,5 eth an o l. Markers were p r e p a r e d from p u r e glyoxylic, p y r u v i c , o x a l o a c e t i c a n d a - k e t o g l u t a r i c acids x*. Bie,ckiTR. Bio~hys. Aaa, 67 0963} ~ 6 - 2 ~
ISOC['FI~ATE
LY.'..%F. A N D
"IRIO.qEPi~|OSPII.;,'rE
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I~I" ~ U L T %
Isocitrate lyase G l y o x y l i c acid f o r m a t i o n w a s verified h v p a p e r c h r o m a t o g r a p h y <)f t h e k e t o a c i d p r o d u c e d . R v v a l u e s of t h e d e r i v a t i v e f r o m the r e a c t i o n m i x t u r e a n d its eel,tar r e a c t i n n w i t h ethane)lie N a O H were t h e .~ame a~ tho.,~e o f a u t h e n t i c gl.voxy]l¢ a c i d 2,4-dini iroplwtlv l h y ( l r a z o n e . -,-t,^. ,,. ~ c c t r u m .,,"¢ +~ ..... •. . ~.,.,...,,,' . . .........,. ;F.:St.÷;sVi;cz:'.qhydrazone. . c o n f i r m c . l the i d e n t i t y o f t h e k e t o acid. I t .~howed a m a x i m u m at 4So[tilL in a q u e o u s alkali, while tile r.~tio o f t h e a b s o r b a n c i e s a t 49o m/x a n d 540 ntlt was 1.84. T h e s p e c t r a ot ttle p y r u v i c a n d a . k e t t ) g h t t a r i c a c i d derivativt2.-..show a ph~tcau b e t w e e n these w a v t : l e n g t h s , w i t h a r a t i o a p i ) r o x i m a t i n g unit3:~, while a u t h e n t i c g l y o x y l i c acid d i n i t r o ~ , h e n y l h v d r a z t , n e h a s a r a t i o o f t. o. T h e m:mi-sl'mcific a n a l y t i c a l nl~,thod o f ~|Z'FADI.~EN ANt) HO~#,°ESte ()ft'('r~ f u r t h e r e v i d e n c e for g l y o x y t i c acid f o r m a t i o n , as it is spte.cific f~r aldeilyde.s t~. T h e s p e c t r u m o f t h e d e r i v a t i v e f o r m e d f r o m a u t h e n t i c glv~)xylic acid wa~ t h e s a m e as t h a t f o r m e d f r o m t h e r e a c t i o n p r o d u c t , s h o w i n g a niaxi~num ~lt 520 in/t~. T h e r e v e r . ~ r c a c i i o n o f f ~ r m a t i o t l o f i.-xwitric acid I'r~m g l y o x v h c at'id ~tnd s u c c i n i c a c i d w a s r e a d i l y d e m a n ~ t r a t e d 2, t h o u g h c h r o m a t o g r , t p ] ~ y failed to d e t e c t s u c c i n i c a c i d a f t e r t h e i s o c i t r a t e l y a s e reaction, it s e e m s r e a s o n a b l e to c o n c l u d e t h a t suc.cinic acid w a s f o r m e d d u r i n g r e a t t i o n a n d f u r t h e r m e t a b o l i s e d b v tile u n p u r i f i e d extract. I.-mci~.rate l y a s c a c t i v i t y o f u n p u r i f i e d extract.~ was c o m p a r a b l e t o t h a t relx)rted p r e v i o u s l y for s i m i l a r e x t r a c t s o f a c e t a t e - I z r o w n A. v i n e l a n d i i a n d A. agile t a n d o f P s e u d o m c m a s a e , u g z n o s a 2. T h e hibIhcst a ( ' t i v i t v o b t a i n e d xva.~ r e #mole.- t~f g l y o x v l i c acid p r o d u c e d p e r m g of p r o t e i n lXJr h, t h o u g h a~:ii,'~t~, d e c l i n e d slig|ltl~, ~-ith s t o r a g e . A f t e r 6 m n n t h s a t - - z o ~ in o.0z 5 M p h ~ s p h a t o buffer ( p H 7 o ) ceil-free e ~ t r a c t s r e t a i n e d 75~/0 o f t h e i r initial a c t i v i t v . Sucro.~e-grown cells s h o w e d negligable isoc i t r a t e lya.~e a c t i v i t y . Actl,mtio~z ~.4th ¢ y s t e i n e a n d E D T A T h e a c t i v a t i o n o f A z o t o b a c t e r i s o c i t r a t e l y a s e b y a r a n g e o f cv.~teine or E D T A c o n c e n t r a t i o n s is sh(Jwn in Fig. x. C.wteine is less effective t h a n E D T A . tn~th in e
~ X ~ r - (tYrA
oct~0r/3r~ Fig. I. The effect of cvs*.einc and ED'r. ~, on i,.~>cit~ate lyase acti~'ity of .4. vi~..la.dii $ ~xtracts.
E ~ h Thulthcrg tul0t- contaz~ned (in i~molesJ3.o ml final volume) : Trts buff~ar {pH 7.f>) zoo; MgCI=, (): ~='~DTAor neL~traliscO ~:y~teine hvclroCltlorit!c. ~ intlk:~ttcd; N~-oL-l.cdxaitratc. i n nit|c-arm. ,o; plus Aze~tobacter extract, l.o rnl~_ ' Reaction ~wL~ for 23 rain at 3o" after 5 rnin preincobation. Biechim. ~i¢~.hv~ j- . ° Acla, 67 {1963} ~zO--z39
I. R. K R N N E . D Y . M. J. D I L W O R T H
230
t e r m ~ o f c o n c e n t r a t i o n r e q u i r e d , a n d in m a x i m u m a c t J , , i t y r e a c h e d , T h e r a p i d d e c r e a s e in lya-~e a c t i v i t y a b o v e 2 m M E D T A is a p p a r e n t l y d u e t o i n h i b i t i o n cart -~d b y c h e l a t i o n o f Mg ~ . T o e s t a b l i s h t h e c o n c e n t r a t i o n o f M g t+ r e q u i r e d to m a i n t a i n m a x i m u m e n z y m e a c t i v i t y in t h e p r e s e n c e o f o.6 7 m M E D T A ( v ' h i c h w a s , , m p l e for a c t i v a t i o n ) , a s a t u r a t i o n c u r v e for Mg ~÷ w'a-~ d e t e r m i n e d . Since i ~ c i t r a t e lya.~e a c t i v i t y w a s o n l y s l i g h t l y b e l o w m a x i m u m in t h e p r ~ e n c e o f eqlximolar E D T A a n d Mg z~, ~ufficicnt Mg a* in s u b s e q u e n t e n z y m e a . ~ a y s w a s e n s u r e d b y use o f a 3-fold excess. A s c a n he .seen f r o m Fig. 2, t h i s d i d n u t i n t e r f e r e with the activating influence of EDTA.
3I
41'
.$/'
o
4
~,
~er~o e 5 ~ 1 ~ tml
Fig. 2. Isocitrate lyasc activation by Mg t-. Thunberg tubes contained reagents described under l~tg÷ x, except: A. p~tt."landii extract. 0. 5 rag; EDTA. z/~rnotes: MgCit, total am indicated, in fit~i volume of 3.o nd, Reaction was for x.$ rain ~t 30 ° ~J,fter 5 rain preingubation.
A d d i t i o n nf c y s t e i n e t o e x t r a c t m a x i m a l l y a c t i v a t e d w~th E D T A d i d n o t r e s u l t in increa,~ed a c t i v i t y ; in f a c t , l o w e r a c t i v i t y w a s r e c o r d e d . A c t i v a t i o n b y E D T A w a s a p p a r e n t l y r a p i d , for t u b e s w e r e i n c u b a t e d ':'or o n l y 5 ntin b e f o r e r e a c t i o n w a s s t a r t e d . I n c u b a t i o n o f e x t r a c t v d t h E D T A in t h e p r e s e n c e o f ,Mg~÷ in T r i s b u f f e r for p e r i o d s u p t o 6 h b e f o r e s t a r t i n g r e a c t i o n d i d n o t r e s u l t in a n y s i g n i f i c a n t c h a n g e in a c t i v i t y . T h e p a t t e r n o f a c t i v a t i o n o f A z o t o b a c t e r isocitl~ate l y a s e d i d n o t c h a n g e w i t h t i m e , E D T A still b e i n g s u p e r i o r t o c y s t e i n e i n a c t i v a t i o n a f t e r 6 - m o n t h s ' s t o r a g e at - - 20% T h e m e c h a n i s m o f b o t h E D T A a n d c y s t e i n e a c t i v a t i o n w a s a p p a r e n t l y t h e ,~-ame. W h e n p r o t e i n c o n c e n t r a t i o n w a s d e c r e a s e d f r o m x.o r a g / 3 m i t o o . x 2 r a g / 3 m l a n d E D T A c o n c e n t r a t i o n r e d u c e d t o 0.067 raM, i s o e i t r a t e l y a s e a c t i v i t y wa_~ o n l y 7 4 % 'ff t h a t o b t a i n e d w i t h 0.(> 7 m M E D T A . T h i s i n d i c a t e s t h a t E D T A a c t i v a t i o n w a s d e p e n d e n t o n its final c o n c e n t r a t i o n in t h e a ~ s a y m e d i u m , a n d n o t on a s t o i c h i o m e t r i c r e l a t i o n s h i p x~'ith t h e a m o u n t o f p r o t e i n .
zffect oj p n T h e e f f e c t ot pVi on J s o c i t r a t e l y a s e a c t i v i t y is sho~na in Fig. 3- M a x i m u m •, ~ v : : y ~ c e u r r e d b e t w e e n p H 7.6 a n d 8.o in n.o66 M T r i s b u f f e r . T h i s c o r r e s p o n d s c l o ~ l y t o ~he o p t i m u m o f p H 7.7 o b s e r v e d w i t h a n e x t r a c t f r o m P s e u d o m o n a s im~igofo'ais. a/oeAim. B/o-phys. Aaa. 67 (t963) 2a6.--239
IS(K'ITI~'~TE I.VASE AND TRIOSEPHOSPI~A-Vt,; I)HHYDROGENASI.:
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.__.J
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Fig. 3- p H depertdence_ of i~;0eitrate lya~e. "l'htznbtrr~ tubes contained (in ~umoIc.~/.~.om! final volume}; TI~.s buffer. -'tin; .3,IgCll, (~; EDTA, 2; ,Na~-~L-tsocttrat~. -<~; t;lu~ .'l. t'i~::'!an,t~i ex.*-ract, t.tJ mg~ I~.eaction wa.~ for Io miu itt .to° aftor 5 rain preincuhatiozt.
Protein concentration
When sufficient EDTA or cy~teine was added to achieve maximum activity of isocitrate lyase, glyoxylic acid formation with varying levels of Azotobacter extract w a s a s s h o w n i n F i g . 4. T h e p l o t for t h e s e r i e s w i t h E D T A a c t i v a t i o n is q u i t e l i n e a r a n d , u n l i k e t h a t with cysteine, passes thr~mgh the origin on extrapolation. At low protein cnnccn-
0.1
0.2 0.3 O.,a 0_5 nng ~c~erio~ proteir.
0.6
Fig. 4- 1-e~itrate ly~qe a c t i v i t y with increasing ct,;~centration of Azotobacter extract. Thunlx.rg t u b ~ c n n t a i n e d {in t=moles]3.o ml final volume~ : Tris bttffcr (pff 7.8}, zoo; Ms~('lz, 6; N,~-~LLsocttr~te, in side-arm, i5; ~"9---(). E D T A z; H . cystcine hydro~hloridc, t 5, R~-action was c o n t i n u e d for =-z rain a t 3o" after 15 mid p r e i n c u h a t i o n with .4. a,iT:¢landii extract added as inrlic;tbe~I. t r a t i o n s , c y s t e i n c h a s o c c a s i o n a l l y p r o v e d s l i g h t l y s u l ~ , , i o r t o E D T A (see T a b l e I l L s u g g e s t i n g t h a t u n d e r t h e s e c o n d i t i o n : ~ it e x e r t s s o m e u n d e f i n e d a c t i v a t i o n . I n c o m pa_,ison t o t h e E D T , ~ e f f e c t a t h i g h e r p r o t e i n l e v e l s ( F i g . 4), s u c h a n e f f e c t a p p e a r s to be very slight. B~ocklm. Biop~ys. Aa~. 67 (t963) zz~>-z39
232
|÷ R. KENNEDY. M. J, DILWORTH
Glyovvlate reaction w i t h thiol.s T h e depre.-sion o f i s o ¢ i t r a t e lya.-a.~ a c t i v i t y x~-ith h i g h e r l e v e l s o f c y s t e i n e (Fig. l) w a s itnlikclv to be cau.,~ed b y i n h i b i t i o n c o n . , ~ q u e n t u p o n M g t-~ b i n d i n g , as o c c u r r e d w i t h E I ) T A , for c y , t e i n e ha~ o n l y low a f f i n i t y fc~r Mg 2+ (log K less t h a n 4) t~. I n a d d i t i o n , t h e R~wcr a c t i v i t y :~bserved d u r i n g a c t i v a t i o n w i t h E D T A p l ~ cy.,.teine. r a t h e r t h a n E D T A alone, s u , . ~ e s t e d ~ o m e i n t e r f e r e n c e b y cy~teine. R e a c t i o n b e t w e e n cvsteint- a n d g l y o x y l i c a c i d is n o w well s u b s t a n t i a t e d *~,*~, a n d w e I~ave also o b s e r v e d rapid reaction between glyoxylic acid and cysteine at neutral pH. G l y o x y l i c acid c o u l d not b e detec.tt~i a f t e r a 1.5 m M s o l u t i o n w a s i n c u b a t e d for 30 rain at 3 o" w i t h a x6 m M s o l u t i o n o f c y s t e i n e in 67 m M T r i s b ~ f f e r ( p H 7.8) w h e n th:~ ~tnaivticai m e t h o d o f FI~IEDEI~A-~.TM ~,NI~ I-[AL'GEN~-~ w a s u~ed ( w i t h o u t s o l v e n t e x t r a c t i o n ) . \~'ith t h e m e t h o d o f McFA[)D~':y At~D HOWES ~*, t h e r e c o v e r y o f g l y o x y l i c acid w i t h h e a t i n g t i m e in p h e n y i h y d r a z i n e h y d r o c h l o r i d e at p H ~ wa~ as s h o w n in Fig. 5- T h e c o n t r o l series a l s o s h o w n in Fig. 5 c o n t a i n e d n o c y s t e i n e .
o
5
~3
T i r r ~ o f tte Qtin~ (rain)
15
F;g. 5- (~l>cTx)~'i~ a,:td re~:overy from ryst~-inu complux with heating titan. C,|yoxylatc was incubated at 3°" f(~r 30 mia in soluuon containing (in ~anolcslO.o roll: Tri~ bufft-r (pH 7.8) xooo; EDTA, 4 ; cvst~mc hydrochlot3tte, loo; ~lyoxylic acid. 9. A control contained no ~yst~inc. Colour proctuccd ~va_~ as shOWl~ wh~rn o.Z'05 ml sarnple~ were analyf~ett with pheny~hydrazinc hydrochlo:idc ~* by heating for vafiab],, lime at 95"'- G ..'~, plus cysteine; O---O, control. G r e a t e s t col()ur d e v e h ) l ) m e n t wa~ o b t a i n e d w h e r e h e a t i n g w a s o m i t t e d a n d t h e s a m p l e i n c u b a t e d w i t h p h e n y l l w d r a z i n e h y d r o e h l o r i d e a t r o o m t e m p e r a t u r e for 2o rain. T h i s e m p h a s i z e s t h e n e e d fu, ~:a~eful : 4 t a n d a r d i s a t i o n o f h e a t i n g t i m e , or i t s e l ) r e p l e t e o m i s s i o n , in a ~ r e e m e n t w i t h t h e c o n c l u s i o n s o f KRAM "R gl~ al. ~7 for a m e t h o d u~ing e s s e n t i a l l y t h e s a m e p r i n c i p l e . T h o u g h it is o b v i o u s t h a t h e a t i n g r e l e a s e s g l y o x y l i ¢ a c i d f r o m its c o m p l e x w i t h c y s t e i n c , t h e r a t e o f d e c o m p o s i t i o n o f g ! y o x y l i e acid p h e n y l h y d r a z o n e t o y i e l d a p r o d u c t w h i c h is n o t o × i d i s e d t o t h e f o r m a z a n m a k e s t h i s m e t h o d i m p r a c t i c a l f l e for t h e d e t e r m i n a t i o n o f g l y o x y l i c a c i d after incubation with cysteine. P e r c e n t a g e loss o f x m.~! g l y o x y l i c a c i d i n c u b a t e d witlt t h e s a , . c r a n g e o f c y s t e i n c c o n c e n t r a t i o n s a s u s e d for a c t i v a t i o n o f ist~citrate l y a s e (Fig, x) i.~ s h o w n in T a b l e I, T h e s e r e s u l t s s u g g e s t t h a t c y s t e i n e , a t s u f f i c i e n t l y h i g h c o n c e n t r a t i o n s , is a.s c o m p l e t e a n a c t i v a t o r as E D T A , a n d t h a t d i s c r e p a n c i ~ b e t w e e n t h e t w o r ~ u l t f r o m i n t e r action between glyoxylic acid and cysteine. 13iochim. 13iophy~. A ¢tu, t>7 (:963) 2~(>-239
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C o r x d i t i o n s o f a s s a y : ~ m+xl g l y t ~ x y l i c a c i d w a ~ i n c u bat~:ti .,it 3'~ , v i t h c y s t , - i , , , ' f , r -':~ m~.n i n 2 0 0 m.M T r i s b u f f e r {pP: 7A/.); z n , M S l g t ' t ~ ; c ~ b 7 m.'Xt E I ) ' I + . k . li~,al ".+~)!+,.,r~ .j+.u. ml t'glyc,xyl,~te in o. 2 4 - m l ~• d t l I l l l h + ~ "~,'~.~'i a q : ; a v o d
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~.Vhen g l u t a t h i o n e was te~ted fi)r interaction with g l y o x y t i c a~id in the syst~,m.~ described above, n o interfering effect coulcl be found. G l y o x y l i c acid recovery with z , 4 - d i n i t r o p h e n y l h y d r a z i n e ~ or p h e n y l h y d r a z i n e '~- was the ~atne as that in ccmtrol m i x t u r e s to w h i c h no glutathiom: had been added. ( ; l u t a t h i o n e has been reported to react w i t h c a r b o h ; l s ~ ; however, a n y c o m p o u n d formed here m u s t h a v e been easily disbociable. A clivity with time S p e c t r o p h 0 t o m e t r i c ag~lx' h,cthud.~ fi,r i~,. itr.~:;- l y a . ~ d e p e n ~ a n t on tile fl;rmalion o f c o m p o u n d s o f g l y o x y l i c acid with semicarbazide (~i;~,~rhing at z52 mt~. see ref. 24), and with p h e n y l - h y d r a z i n e hydrcwhioridc (absorbing at 324 m/z, see ref. 25), s h o w short lag periods before significant changes in ahsorbancv occur, probably due to slow reaction betw~en g l y o x y l a t e and the reagentg. W i t h A z . t o b a c t e r e n z y m e , and with direct chemical assay of g l y o x y l i c acid formation, the time course of i~)citrate lyase reaction v'r~ as shown in Fig. 6. The line o f best fit, b y the m e t h o d of least-squares, pas_c.es almost directly through the origd n + 1 .~,[
g E
b G L~
S C
&, ~) R,ea~--tk>n t i m e
~
15
(mll~)
F i g . 6+ I A n e a r i t y o f i s c ~ i t r a t e I)'a.~e r e a c t a o n w t t h t i m e . ~ V a r b u r g fla-~ks c o n t a i n e d (in l, m o l e ~ / 3 - 0 m l final r o t , . :,e) r "l'ris b u f f e r ( p H 7-6) 2 ~ ; MgCI=+ 6 : E D T A . z : N a = - D t . - i s o c i t r a t c {.~idt~-arrlt). 3 o ; p l u s A. uinetandB o~tt.rat:t, o+7,q m g . R e a c t i o n w a s s t a ~ e c i afte= a'" , n i n p r c i n c u b a t i o n a t 3 0 ° and stopped at the times indicated.
Bio~hi,n. Biopliya. +4tta, 67 ( t q 6 3 ) 2 2 6 - 2 3 9
234
I. It. K E N N E l ) V , M. J . DILWORTH
The effect of stcbstrate concentration Th e s u h s t r a t e of P. i~digofera i~ocitrate ly~se ha_~ been r e p o r t e d as I)B(+)isocitrate ts, t h o u g h o t h e r workers 2,s cons i de red the l.B(+)-form as t h e act i ve isomer for i,~citrate lyase from o t h e r microbes. DL-Isocitric acid was e x a c t l y h a l f as a c t i v e as the single i~omer fi>r isocitrate lya.ge, indicating t h a t onl y one form was m e t a bolised and confirming t h a t t h e i n a c t i v e fiwm p r o d u c e d no inhibitionS, a*. In this p a p e r we h av e followed t he n o m e n c l a t u r e o f VtCKERV za. who r e c e n t l y suggested an u n e q u iv o cal system to resolve t he present confitsed position. T h e E n z y m e Comnfission has r e c o m m e n d e d t h a t isocitrate lyase (form erl y isocitritase) be k n o w n s y s t e m a t i c a l l y as L~-isocttrate g l y o x y l a t e lyase. This should be superseded b y t hreol)s-i~x:itrate glyox_vlate lyase to agree w i t h t h e n o m e n c l a t u r e o f VICKERY tB for t h e n a t u r a l i.-~citric acids. T h e reaction rate c u r v e for s a t u r a t i o n o f A . vinelandii isocitrate lyase w i t h thre~-~>=-isr~citrate is shown in Fig. 7. a nd c o n f o r m s with ;~ICHAELIS--}IENTE.N"
i azl
1C/~
"~'--e'------------e
rrlM I'nreo -C~,- is,o c i t r o t o
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Fig. 7- E v a l u a t t o n of K m for i s o c t t r a t e lyase. " l ' h u n b e r g t u b e s c o n t a i n e d {in # m o l e s / b . o md final v o l u m e ) : "rris b u f f e r ( p l l 7-b) 400; MgL'It, t 2 ; E D T A , 4 ; t h r e o - o = - i s o c i t r a t e (as N a = - o L - f o r m i n s i d e - t t r m ) , as i n d i c a t e d ; p l u s .4. vi~ela~dii extr~vct, o - ] 5 m g p r o t e i n . T u b e s w e r e l ~ r e i n e u b a t e d &t 3o ~ for xo rain a n d t h e r e a c t i o n s t o p p e d a t 7 rain b y a d d i t i o n o f o . 4 ml o f 8o°/0 t r i c h l o r o a c e t i ¢ a c i d . R e s u l t s s h o w n a r e s i n g l e a n a l y ~ e s o n d u p l i c a t e d l e v e l s ot" s u b s t r a ~ .
kinetics. A LIXEW~:Avr:R--BuRK=¢ d o u b l e reciprocal ph)t of t h e r a t e of g l y o x y l i c acid forrr, atic;n r:cr,~ur, t h e thrc~, :;o-isocitlaL~: c , , n c e n t r a t i o n was linear, a n d e x t r a pointed to a Km o f 8.6- zo--" M. This lies within t h e r a n g e o f Km values p r e v i o u s l y r e p o r t e d o f 4.5" xo-~ M (see rcf. 2) a n d 5" zo-s M (see ref. 28). Purified y e a s t e n z y m e required t . 2 . xo" s M "I.s( + ) - i s o c i t r a t e ' " for h a l f - m a x i m u m a c t i v i t y s. A r e p o r t t h a t isocitrate lyase from P. indigofera was i nhi bi t ed b y c o n c e n t r a t i o n s of "l)0( i-}-isocitrate" g r e a t e r t h a n 8 . x o - 4 M , or of DL-isocitrate of x . 6 - x o - a b l (.gee ref. I8). was not in a g r e e m e n t with the present results. Because it was at first s u s l ~ c t e d t h a t this i nhi bi t i on was t h e result o f thiol r e a c t i o n with g l y o x y l i c acid a t higher cxmcentratinns of isocitratc, a nd he n ce o f g l y o x y l a t e , r a t e s o f r e a c t i o n ~Sth e i t h e r cy s tein e or E D T A as a c t i v a t o r s were followed. At low p r o t e i n c o n c e n t r a t i o n (4o p4g/ml) w i t h low g)yoxylic acid p r o d u c t i o n , a c t i v a t i o n b y c y s t e i n e a n d E D T A wa_~ closely similar. As shown in T a b l e II, no inhibition was o b s e r v e d at c o n e e n t r a Bioc~im B i o ~ s . Acta, 67 (t~3) 2=6--z39
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Conditions of as,gay were :ts d.r~ribed in Fig. 7 ~-xccpt: .-I ~,inela~ldti extract, 0.-- 4 mg vr~,teia; reaction was for ~z rain m 6.o-m! t-:rml volume. G;;mr'¢l:'g :~r ~:~ [.,~me,! (~.m,cdtJ ~~ * ' )
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t i o n s o f u L - i c ~ 3 c i t r a t e u p t o 8 . s o a M. C l e a r l y +'.,r~ i ; i h i b ; t l o n o b s e r v e d In is n o t d u e t o a r e a c t i o n o f t h i s t y p e , e s p e c i M l y ~ g h t t a t h i v n e w a~ u s e d f o r a c t i v a t i o n . I t t h e r e f o r e a p p e a r s t h a t s u b s t r a t e i n h i b i t i o n is n o t a g e n e r a l p r o p e r t y o f i s o c i t , ' a t e lya.~e.
T r i o s e p h o ~ p h a t e dehydroge~rase T h e p r e s e n c e o f t h i s e n z y m e i n A . ~'inda, d i i h a~ b e e n i n f e r r e x t f r o m i n d i r e c t e v i d e n c e b y M o R ' r E ~ S O N , H.~,HtLTO.~ A.~r> WtL.~O.', m. A l t h o u g h d i r e c t r e d u c t i o n o f N A D d i d n o t o c c u r in a n e x t r a c t w i t h a d d e d f r u c t o s e d i p h o . ~ p h a t e a n d a l d o l a s e
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ltiochi,n. Biophys. .4cJa, 67 (I963) z26--z39
236
I. R. KENNEDY, M. J. DILSVORTH
(IsC 4. I.2.7), N A D H 2 o x i d a t i o n could be d e m o n s t r a t e d in such systems. S u b s t a n t i a l N A I ) r e d u c t i o n has been a c h i e v e d w i t h p a r t i a l l y purified enz3m~e from A. vinelandii. As shown in Fig. 8, x mbl E D T A was slightly b e t t e r for a c t i v a t i o n of N A D r e d u c t i o n b y g l y c e r a l d e h y d e 3 - p h o s p h a t e and A z o t o b a c t e r e x t r a c t t h a n 3-5 mM cysteine. Sl:.(;,xL ,XXD BO~'ER r h a d n o t e d a similar SUl~riority o t" E D T A over cysteine for a c t i v a t i o n of t r i o s e p h o s p h a t e d e h y d r o g e n a s e from muscle. P y r o p h o s p h a t e g a v e g r e a t e r a c t i v i t y than did Tris of the s am e p H a n d m o l a r i t y (22.5 raM] a n d m a x i m u m a c t i v i t y was o b t a i n e d at p H 9.o. Unlike t h e p r e p a r a t i t m o f St-:t;Al. AXlJ BOYEU 7, Tris buffer did llot allow a l o n g : r pc.clod o f lineal a b s o r b a n e y change. N A D P was not r e d u c e d in t h e s ys t e m . Th e g l y c e r a i d e h y d e 3 - p h o s p h a t e c o n c e n t r a t i t m for h a l f - m a x i m u m react i on velocity wa.~ e~timated er at 5.3" I o- s M a t p H 9.~) and 3o ~. An a t t e m p t was m a d e to a c t i v a t e the e n z y m e with cysteine, a n d t hen to r e m o v e the excess b y dlalv~i~. . _. 2 ml of enzyme. (4.6 m g of protein) were t r e a t e d w i t h 0.0 5 ml " f ~ 2 ~t cvsteine h y d r o c h t o r i d e a t p H 7.z for z h at o ° in a T h u n b e r g t u b e u n d e r nitrogen. T h e e n z y m e was q u i c k l y t r a n s f e r r e d to dialysis t u b i n g ( E D T A washed) and dialysed x~-ith stirring against x 1 of deionised w a t e r in an a n a e r o b i c F i l m e r j ar filled xwith nitrogen and c o n t a i n i , g an o x y g e n a b s o r b e n t . A f t e r x8 h dialysis, tlie e n z y m e was r e m o v e d a nd a,~sayed withol, t eyste.ine. No increase in a c t i v i t y a b o v e t h e u n t r e a t e d e n z y m e could be d e m o n s t r a t e d , a l t h o u g h t h e dialy.',;cwl e n z y m e coul d be r e a c t i v a t e d b y c.x,steine to an a c t i v i t y identical with t h a t f o u n d a f t e r t he initial cvsteine activ atio n. This result indicates t h a t t h e r e is a r e q u i r e m e n t for e o m p l e x i n g r e a g e n t in t h e m e d i u m during e n z y m e action, and poi nt s to the possibility t h a t the a c t i v a t i o n was c o n c e r n e d with a p r o t e c t i v e effect o f cy~teine ag,tin~t o t h e r c o m p o n e n t s o f t h e reaction m i x t u r e . DI.qCt;.q.qION In a g r e e m e n t with previous findings (see KOI~NI~I.'.HG~ for review), i.~mitrate lyase has again p r o v e d a d a p t i v e to g r o w t h on acetate. During g r o w t h on .sucr.~e. which tl()es not require net synthesis of d i c a r b o x v l i c acid.;, no isocitrate lya-.;e a c t i v i t y was .'letccted. T h e finding t h a t E D T A is an efficient a c t i v a t o r ,)f i s . c i t r a t e lyase assumes Slx~cial signilicance because of the serious i n t e r f e r e n c e in ashtray b y cysteine. Reactions of t h e t y p e occurring b e t w e e n g l y o x y l i c acid and cysteine are now well docum e n t e d . T h e ease of reactio:~ b e t w e e n c y s t e i n e a n d k e t o n e s at n e u t r a l p H a n d r~×)m t e m p e r a t u r e h as been noted'~L G l y o x y l i c acid a n d cysteine yield a s u b s t a n c e which o n l y r eacts wit|. p h e n y l h y d r a z i n e at m o °, a n d form s a thiazolidine qui t e different f r o m t h e n o r m a l h y d r a z o n e f o r m e d in t he absence of c y s t e i n e % t h u s explaining inc o m p l e t e recoveries of g l y o x y l a t e . T h e seriou~sne~s of the react i on b e t w e e n g l y o x y l i c acid an d cysteine has r e c e n t l y been r e l a t e d t o t h e assay of i s o c i t r a t e lyasetL T h e f o r m a t i o n o f a c y s t e i n e - g l y o x y l a t e c o m p l e x m a y be c o n s i d e r e d t o p r o d u c e t w o results: (a) an a p p a r e n t lowering o f a c t i v i t y d e p e n d e n t on r e c o v e r y of g l y o x y l a t e , w h i c h will be a f u n c t i o n of t h e time ~f e n z y m e assay used, and t h e degree of h y d r o lysis in t h e a n a l y t i c a l m e t h o d (a s a t i s f a c t o r y m e t h o d for h y d r o l y s i s of t h e c o m p l e x sb-ould o b v i a t e difficultie.~ ee.u'~--d b y its f o r m a t i o n t t ) ; ( b ) a lowering of a c t i v i t y d-~pending on the e x t e n t o f r e m o v a l o f cysteil~e b y g l y o x y l a t e . D uri ng t h e course o f B i o c A i m . B~opky.~. Ac/a. 67 (t9~$) 2 z 6 - 2 3 9
ISOCITRA'rE
].VASE
AND
TRIOSEPHO-'gPi!AI'E
DEH'~'DROGENASE
237
e n ' ~ y m i c r e a c t i o n , a c o n t i n u o u s l y d e c r e a s i n g cy.~teine c o n c e n t r a t i o n t m a y he e x p e c t e d t o m a k e t h e d e t e r m i n a t i o n o f a b s o l u t e a c t i v i t y ditlicult. I t is t h e r e f o r e i n a d v i s a b l e t~ use cy.steine for i s o c i t r a t e l v a s e a c t i v a t i o n . G l u t a t h i o n e is a p p a r e n t l y a s u i t a b l e s u b s t i t u t e , b u t E D T A h ~ t h e a d v a n t a g e t~t" ...tAt n ~ t b e i n g a ~ t o x i d i s a b i e , a n d .N.tL~...... thereful~, aihJw a e r o b i c a s s a y , f i~ocitrate lyase. T h e i m p l i c i t a s s u m p t i o n in cv~tein,: a n d g l u t a t h i o n e a c t i v a t i o n o f i ~ o c i t r a t e lya~e h a s l~een a specific effect o f t h e i r -.%H g r o u p cm t h e e n z y m e m~flec~]Ie. [3} c t m t r a s t , E D ' F A d o e s n o t pos.~c~ .inch a g r o u p . It w o u l d t h e r e f o r e appe~tr t h a t exp l a n a t i o n s i n v o l v i n g cy.~teine in r e d u c t i o n of e n z y m i c - S H grlmups a r e improl~al~le in c o m p a r i s o n w i t h o n e i n v o l v i n g b i n d i n g o f h c a x ' y - n t e t a l ions f r o m t h e etnzyme. T h e n o n - a d d i t i v i t y - f t h e c y s t e i n e a n d E I ) T A effe~t~ is s u p i , o r t i n g e v i d e n c e t't~r t h e similarity of their action. S t i m t t l a t i o n b y m e t a l - c h e l a t i n g s u b s t a n c ( ~ h~t~ b e e n sho~vn f,~r a widt: r a n g e or" enzyme_~. D l ; P a s e fEC 3.,q.2.i) is activatetq h'~, 2 , 2 ' - d i p y o d y l a2, K ~ - a ( : t i v a t e d a l d e h y d e dehy(lrt~gena~e (EC 1.2. I . 5 | l)v t h i ~ l ~ , h i s t i d i n e a n d EI)T:X ~. a n d ~'isa c o r d t i c decarboxyla_,~, (EC 4 . I . i . 6 ) b y c:.-.~t~,ino, E D T A a n d 8-hydr,~xx'quinoli~te '~. C h e l a t i o n o f i n h i b i t o r y m e t a l s h a s b e e n s u g g e s t e d as t h e m e c h a n i s m o f a c t i v a t i o n b y E D T A o f m u s c l e t r i o s e p h o s p h a t e dehvdragc~a_¢,e (EC 1.2.~.[2) ~. heart-mu.~:l,o x i d a s e p , ' e p a r a t i o n s as a n d p h o s p h o g h ~ c o r n u t a . c e (I"C 2.7.5. I ) ~ . a L P u r i f i e d y e a s t i . ~ c i t r a t e l y a s e ,.'~ i n h i b i t e d b y low c o n c e n t r a t i o n s o f Cu ~-'. ( ' d ~a n d Zniv0 w h i l e s u b s t a n t i a l i n h i b i t i o n o f isocntra:e l.x'ase t m c u r r e d in lo -r .X[ p c h l o r o m e r c u r i b e n z o a t e , a n d this i n h i b i t i o n w a s 8o';.~ rever.~ed b y ex~:e~s cvstei~te ~. Tiffs p o i n t s to t h e pL'u>ence o f an e s s e n t i a l - S H g r o u p in t h e y e a s t e n z y m e , a n d it is t h e r e f o r e l i k e l y t h a t a c t i v a t i o n . f A z o t o b a c t e r ettz)'rne b y c y s t e i n e a n d E D T A is t h e r e s u l t o f c h e l a t i o n o f i n h i b i t o r y h e a v y m e t a l s b o u n d t,~ it, p e r h a p s to a n .%I1 g r o u p . T h e p o ~ i b i l i t y o f o t h e r m e t a l - b i n d i n g s i t e s r e m a i n s : itt.s'rv,'~N az p r e s e n t s e v i d e n c e t h a t Z n =" i n h i b i t i o n o f phosphoglu~~m~utiL~e is n o t d u e to t h e f o r m a t i o n o f a .-ilercaptide a n d s u g g e s t s a l t e r n a t i v e b i n d i n g s i t e . ~ - - a m i n o , intidazoly[, t'nrbt~xvI and phosphate. T h e c o n c e n t r a t i o n or" c y ~ t e i n e recluir~"l t o a c t i v a t e i s o c i t r a t e lya~e in A z o t ~ b a c t e r e x t r a c t s is c o n d i d e r a b l y g r e a t e r t h a n t h a t o f E D T A . Allowing G>r i n t e r a c t i o n o f g l y o x y l i c a c i d w i t h c y s t e i n e , t h e con~centrath~II t~f t h e thio] r e q u i r e d i~ a t l e a s t 3 raM, w h i l e w i t h E D T A , o.z'.: m M pr~Xucc:~ a l m o s t m a x i m u m a c t i v i t y . I f a m e t a l ion is re_~ixmsible for t h e inactivt" fi>rrn o f i s o c i t r a t e lyase, it appeztn's t h a t cy~t,-ine is less efficient in b i n d i n g t h i s m e t a l . F r o m t h e s t a b i l i t y c o n s t a n t s for c o m p l e x e ~ b e t ~ ' e e n v a r i o u s m e t a l ion~ a n d e i t h e r c y s t e i n e or E D T A t~, c a l c u l a t i o n s w e r e m a d e o f t h e r a t i o o f free to b o u n d m e t a l for a r a n g e o f ions, m a k i n g a l l o w a n c e for f o r m a t i o n o f z : 2 c h e l a t e s betw<~n s o m e m e t a l s a n d c y s t e i n e . F o r o . I 6 m M l i g a n d {the E D T A c o n c e n t r a t i o n g i v i n g m a x i m u m a c t i v i t y , see Fig. z) a n d a contstant m e t a l - i e m level, t h e equilibrium c o n c e n t r a t i o n o f free Co -~~, Fe ~', .~i a-, PU ~ , Z n a~. Mg ~" atnd Mn ~+ w o u l d b e g r e a t e r w i t h c_vsteine tlnan ~ ' i t h F, D T A , while t'.,te rever~e w o u l d b e t r u e fi~r F e ~- a n d H g * ' . I t is therefi~re u n l i k e l y t h a t t h e l a t t e r ioais a r e r e s p o n s i b l e f~>r t h e i n a c t i v e f o r m o f i s o c i t r a t e l y a s e O f t h e f o r m e r , Mg 2¢ , M n 2", Fe"" anti ( ' o z ~ h a v e b e e n s h o w n t o a c t i v a t e i s o c i t r a t e lyaseS, ~.m, m a k i n g it u n l i k e l y t h a t t h e y a r e c o n c e r n e d in its i n a c t i v a t i o n . A l t h o u g h a s t a b i l i t y c : m s t a n t for t h e Cd~'-~=ysteine corr~p!ex i~ n o t a v a i l a b l e , it is /3i,J~ h~m. Bi,pA~'s. A*'ta. 67 (:9~,3} ~2f~--~39
I. R. K E N N E D Y ,
z38
M. J . I)ILXVORTH
p r o b a b l y similar t o t h a t fi.~r t he CdZ+--glutathione cnrnpiex, in which a lower proportion o f m e t a l is b o u n d thm~ in the C d ~ + - E D T A complex. I f these idealised chemical d a t a are applicable to the s y s t e m used for isocitrate ly.~e assay, then, of the cases available for c o m p a r i ~ n , the ions m o s t likely to cau.~e i n a c t i v a t i o n are Zn*", Ni a ' , I+b~+ a n d Cd +'. It is ix)ssible t h a t Cu ~+ is also i n v o h ' e d , A l t h o u g h d e p a r t u r e s from i de a l i t y are o b v i o u s (in t e r m s of p H , t e m p e r a t u r e , ionic s t r e n g t h an d o t h e r ions), the differences in ratios of free to l x m n d m e t a l for E D T A and cysteine are so large t h a t some assessment of t h e relative a d v a n t a g e s of t h e e c<>mi~mnds is ~ i b l e . A v a r i a t i o n in rt~Imnse can be e x p e c t e d duri ng a c t i v a t i o n b y different c h e l a t i n g :.ubstances, the a c t i v a t i n g efficiency o f w hi ch will be a f u n c t i o n of t h e t y p e a n d quantitit-'s o f th e c o n t a m i n a n t s present, a n d not necessarily" a c o n s t a n t p r o p e r t y of the en zy me. It is t h e r e f o r e possible t h a t t h e ~ u p e t i - r i t y of E D T A over c y s t e i n e will l l O t alway s be observed, a l t h o u g h in the m a j o r i t y of caa~s considered above, E D T A ~em<>val ,)f metals .~houh] be m o r e c o m p l e t e . ACK
Nowl.]:. Df;EM ENTS
Gri,*eful acknowlcxlgment is m a d e t o Dr. C. A, PARKER for his c o n t i n u o u s e n c o u r a g e ntent a n d to Professor E. J. U.X'DEmWOOD, D i r e c t o r of t h e I n s t i t u t e , for his s u p p o r t . A c k n o w l e d g e m e n t for financial assistance is m a d e to t h e C o m m o n w e a l t h Scientific a n d I n d u s t r i a l Research O r g a n i z a t i o n for s t u d e n t s h i p s , a n d to t h e U n i v e r s i t y o f W e s t er n Au.~tralia for re qearcl, grants. REFERENCES ~r~. A. S.MtTH A~D I. C. GL'.N'S^LL'S, .%'adUrt, x75 ( ] 9 5 5 ) 774-
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22|.
BIOCHIMICA ET BIOPHYSICA ACTA
BBA XZI6 9 ACTIVATION OF ISOCITRATE LYASE DEHYDROGENASE IN AZOTOBACTER
AND TRIOSEPHOSPItATE VINELANDII EXTRACTS
]. R. I,:HI'~NEDY AND M. J. DII.WORTH" Instffute t~f /Igricult,m, University of $VeJtern A u~fralia, Ned,lands {,4 ustralia)
(Received June t9th, t962} SUMMARY [s0citrate ]yase is 20treed in Azolobact, er v i n e l a n d i i during g r o w t h on acetate. Reported requirements of isocitrate ]yase for Mg 2+ a n d cysteine have been confirmed, bu~ E D T A could replace cysteine for activation, a n d did so more efficiently. This o b x ' i a t ~ the serious interference by cysteipe in glyoxylic acid m e a s u r e m e n t . Isocitrate lyase has a h a l f - m a x i m u m velocity at & 6 - z o -6 M threo-I,s-isocitrate a n d is not inhibited b y high DL-isocitrate c o n c e n t r a t i o n (up to 8 raM). Triosephosphate dehydrogenase has been identified in a m m o n i u m s u l p h a t e fi'actions of crude e x t r a c t s of Azotobacter. This e n z y m e is also a c t i v a t e d b y E D T A nlore efficiently t h a n b y cysteine. It is concluded t h a t the~e activations b y cysteine a n d E D T A are the result of binding of inhibitor}' h e a v y metals in the as,say m e d i u m , T h e possible n a t u r e of ."filch metals is discussed. INTRODUCTION l)uring .~tUdics of oxygen t o x i c i t y on e n z y m e s concerned in o x i d a t i v e p a t h w a y s in Azotobacter v i n e l a n d i i , suitable conditions for assay of i-socitrate lyase (Ls-i.~)citrate g l y o x y l a t e lya.~e EC 4- z.3. I) a n d trio~ephosphate d e h y d r o g e n a s e (v-glycera]dehyde 3-phospoate : NAD oxidoreducta~e, EC x , z . ~ . z z ) were sought. lsocitrate lya.~e~ cataly.~e~ the reversible cleavage of isocitric acid to glyoxylic acict and .~,~cc:.ai= ;,,rid. and has been d e m o n s t r a t e d previously in ext,'acts ~f acetategrown A. vinMa,:dtt a n d A . agi/eL Keqtiircmen¢.~ for a d i v a l e n t m e t a i ion a n d thiol c o m p o u n d s for m a x i m a l a c t i v i t y h a v e been reDortedt-4; SMITH AND GUNSALUSt f o u n d t h a t g l u t a t h i o n e was slightly less active t h a n equimolar cysteine in m e e t i n g the l a t t e r requirement. A similar thiol requirement for a c t i v a t i e n of triosephc~phate dehydrogena_se from mammaUa.n systems has been reported6, 4, t h o u g h SEGAL AND I$OVV:R ~ showed t h a t muscle e n z y m e could be a c t i v a t e d successfully b y E D T A in place of cysteine.
" /~rcscnt addre~ : .Microbiology Unit. D e t r i m e n t of Biochem~try, Univer.~ity of Oxford, Oxford ((;ruat B r / t a / n ) . Bi~im.
Bio~kF~. Ac~. 67 ( t ~ 3 ) Z26-z39
I%OCI1-RATE
I.YA~E
AND
TRI()SF.PIIOSPIlA'rE
DEHYDROGENASE
227
B e c a u s e t h i o l c t ) m p o u n d s w o u l d c o m p l i c a t e st,:dies w i t h g a s e o u s o x y g e n , t h e s e finding:, h a v e b e e n e x a m i n e d fiw A z , ) t o b a c c e r e n z y m e s . S e r i o u s d i s a d v a n t a g e s in t h e use o f c y s t e i n e for i s ( ~ i t r a t e ly:t~e a c t i v a t i c m h a v e .been d e m o n s t r a t e d . METHODS
Bacteriological F o r p r e p a r . ' , t i o n ~,f (..:~tri'.t'.t.~ ~:t>rlt,tt,~,tlg i~,~citrate l y a s e , a s t r a i n or" Azotobacter vinelamlii d e s i g n a t e d S w;m u.~tt ~. A. ~,inelandii 0 w a s u . ~ d for p r e p a r m i o n of t r i o ~ e p h o s p h a t e d e h y d r o g e n a t e . C u l t u r e s w e r e prc.-~erved b y v a e t t u m - d r y i n g 9 a n d c a r r i e d o n M6 a g a r m e d i u m 1° for h r o t h in(~;ulati(m. L a r g e r q u a n t i t i e s o f cells for p r e p a r a t i o n o f cell-free e x t r a c t s w e r e g r o w n on a c e t a t e m e d i u m , o f t h e f o l l o w i n g compf~siti(m, lx-r 1: N a a c e t a t e , 20 g ; K H z P O 4, o . t 5 g; N a , , H l ' ( ) s r x 2 Ha(), ~.x 5 g; M g S O , - 7 H2(), o.2 g ; C a S O a - z H , O , 0.05 g; Na.M,u-~ . ,~ ,,03 g; E I ~ T A tron c(mqflex, o.()x 5 g; Na(21. o . r g; C u $ O 4 - 5 H 2 0 , 0. 4 r a g ; M n S O , - 4 H~O. z.o m S ; Z n S O s . 7 1[,O. o.8 m ~ : [[.~BO~, 2. 4 m g ; w i t h t h e e x t r a c t f r o m a u t o c l a v i n g [2. 5 g o f p e e l e d potat,~ in z 5 g of w a t e r f o r 4 ° r a i n a t 5 Lbfinz. S t e r i l i s a t i o n w a s b y h e a t . w i t h t h e p h o s p h a t e s ~terilised s e p a r a t e l y t o p r e v e n t p r e c i p i t a t i o n . I n o uia ~ v e l e 5'~/0 v o l u m e s o f c u l t u r e g r o w n on n i t r o g e n - d e f i c i e n t s u c r o s e m e d i u m (.X122) ( ~ e ref. 8). W i t h a c e t a t e as c a r b o n , o u r c e , a l k a l i w a s p r o d u c e d a n d t h e p H w a s c h e c k e d p e r i o d i c a l l y d u r i n g g r o w t h a n d ret u r n e d t o n e u t r a l b y t h e a d d i t i o n o f ~terile HCI. C u l t u r e ~ w e r e g r o w n in 2o-1 P y r e x j a r s u n d e r fi)r(:e[t a e r a t i o n at c o n t r o l l e d t e m p e r a t u r e . Cells w e r e h a r v e s t e d a f t e r 36 h g r o w t h b y c e n t r i f u g a t i o n w i t h a Ser,;all m(~deI S S - r . w i t h c o n t i n u o u s - f l o w a t t a c h m e n t , a n d .~tored a t - - 2 0 ", Extract_~ c o n t a i n i n g triosepho.c, p h a t e dehy(lrt)gena.se w e r e o b t a i n e d f r o m cells g r o w n o n M22.
Preparation of cell-free extracts Cell-free e x t r a c t s w e r e o b t a i n e d b y a l u m i n a g r i n d i n g o f frozen cells u. Tlne g r o u n d cell m a t e r i a l w a s ~ u s p c n d e d irl o . o r 5 M p h o s p h a t e b u f f e r ( p H 7.0) at a r:lte c o r r e s p o n d i n g to x g w e t w e i g h t o f cells pet 20 m i o f b u f f e r , a n d c e n t r i f u g e d first for xo r a i n a t 4000 × g to r e m o v e a l u m i n a particle, s a n d cell ~tebris, a n d t h e n for x0 rain a t 2o o(~) × g. T h e s u p e r n a t a n t wiL~ u~ed [br e n z y m e a ~ s a y a n d c o n t a i n e d a p p r o x . 3 m g o f p r o t e i n / m l . T h e s e o p e r a t i o n ~ were c a r r i e d o u t a t or b e l o w 3 :~.
Reagents D i s t i l l e d w a t e r u_sed t h r o u g h o u t w a s deioni.~xt , n B i ~ - D e m i n r o l i t resin ( P e r m u t i t Co.) t o a specific c o n d u c t a n c e o f o . x / ~ m h o / c m . T h e a l u m i n a u s e d fl~r p r e p a r a t i o n of cell-free e x t r a c t s w a s A l o x i t e - 6 o o ( T h e C a r b o r u n d u m Co.. M a n c h e s t e r ) , w h i c h h a d b e e n w a s h e d w i t h o.ox M E D T A ( p H 8.o). r i n s e d s e v e r a l tim~'~ w i t h d e i o n i s e d w a t e r . a n d d r i e d a t 8o ¢. B i o c h e m i c a l s w e r e o b t a i n e d f r o m tile fi;Lh)wing .~ources: c r y s t a a l i n e l y s o z y m e a n d o x a l o a c e t i c a c i d , f r o m N u t r i t i o n a l Biochem::-~ds C o r p o r a t i o n ; N A D a n d N A D P f r o m S i g m a C h e m i c a l Co. ; Naa-threo-osr-.s-i.~Jcitrate f r o m F l u k a C h e m i s c h e F a b r i k ; g l y o x y l i e a c i d m o n o h y d r a t e , f r o m L i g h t a n d Co. ; a n d g l y c e r a l d e h y d e r - b r o m i d e 3 - p h o s p h a t e d i o x a n e o x o n i u m s a l t , f r o m C a l i f o r n i a F o u n d a t i o n for B i o c h e m i c a l Researt:h.
Bi~him. BJophy+..-Jan, 67 ([963) 2=6-230
22~;
I. R
KENNEDY,
M. J . DI1.WORTEi
Aualvtical Isocitrate l y a ~ a c t i v i t y was d e t e r m i n e d b y m e a s u r e m e n t o f gl yoxyl i c acid f o r m a t i o n by the m e t h o d o f MCF^DDEN A.~r~ HOWFS aS. w hi ch involves c01orimelry of the x,5-diphenyl-formaz~an carbx, xylic acid f o r m e d on o x i d a t i o n t~f g l y o x y l i c acid l~ht,nylhydrazone with Kol:e{CN)~. T o e ns ure r e p r o d u c i b i l i t y o f results, t i m e of h e a t i ng was carefully .~tandardised at 5 rain. A reference sam pl e of g l y o x y l i c acid m o n o h v d r a t e wa_s 99-5 O¢ 7o pur e b y t i t r i m e t r i c assay xa. DL-Isocitric acid was t)t;°~n pure, as s ho w n by N A D P r e d u c t i o n w i t h a c r u d e l>reparation o f isocitric d e h y d r o g e n a s e f r o m a c e t a t e - g r o w n A . vinelandii. N A D P r e d u c t i o n wa.~ followed at 34 ° m/x in a B e c k m a n - D U s p e c t r o p h o t o m e t e r , with cell c o m p a r t m e n t at .30°. T h e p r o c e d u r e follc~wed t h a t of I{ORNI3ERGla. with o.o6 M Tris buffer (p14 7.5) replacing p h o s p h a t e . In th e initial e x p e r i m e n t s , t h e a s s a y s y s t e m for isocitrate lyase was similar t o t h a t described b y SstrrH AND GI;N.~AIA.:St, c a r r i e d o u t in T h u n b e r g tubes, or in flasks a t t a c h e d to the m a n o m e t e r s o f a VVarburg r e s p i r o m e t e r . T h e final v o l u m e was 3.o ml, ~sith nitrogen as t h e gas p h a s e t h r u u g h o u t . R e a c t i o n was s t a r t e d b y a d d i t i o n of isocitrate f r o m t he side a r m a f t e r t e m p e r a t u r e e q u i l i b r a t i o n at 30°, a n d s t o p p e d with o.2 ml o f 80o/o (w,'v) t r i c hl or oacet i c acid. A ft er p r o t e i n wag r e m o v e d b y eent r i f u g a t i o n , an aliquot of the s u p e r n a t a n t was a n a l y s e d for g l y o x y l i c acid. Controls to which .~ubstrate was adde~l a f t e r t r i c h l o r o a c e t l c acid a d d i t i o n c o n t a i n e d negligible amounts of glyoxyiate. T r i o s e p h o s p h a t e dehydrogena_~e was ;Lssayed b y o b s e r v a t i o n o f N A D r e d u c tion at 34 ° m/~. T h e a.~say s y s t e m follows t h e p r o c e d u r e o f KREESa, which uses c v s t ein e as a c t i v a t o r . T h e p u r i t y of c o m m e r c i a l DL-glyceraidehyde 3-pho.~pbate i -bromide d i o x a n e o x o n i u m .,~alt was d e t e r m i n e d with A z o t o h a c t e r e n z y m e ; it a.~sayed at b7%. "l'rioscphosphate d e h y d r o g e n a s e was p r e p a r e d from 6 g wet weight of sucrosegl o w n cells, which were g r o u n d for eo min with t5 g o f alumina. T h e g r o u n d m a t e r i a l was take~ up in 48 rnl of 0.03 M p y r o p h o s p h a t c buffer (pH 8.7), c o n t a i n i n g 2.5 ml of o.o~ .',1 n i c o t i n a m i d e , .,,,,--' centrifugtxi for x2 rain at 7ooo x g. E n z y m e was p a r t ially purified from t h e s u p e r n a t a n t b y a m m o n i u m s u l p h a t e f r a c t i o n a t i a n , mc~t a c t i v i t y being p r e c i p i t a t e d b e t w e e n 0.6 and 0,8 ~ t u r a t i o n , T h e e n z y m e was stable f~)r at leagt 3 month.~ at zo °. F u r t h e r purification was n o t u n d e r t a k e n , ~iPce a c t i v i t y could be satisfactt~rily d e t e r m i n e d w i t h o u t o b s e r v a b l e r e o x i d a t i o n of NAt)H.. Pro, to'in wag m*,ngllrod b~' th~ methexl o f I.OWRV el al. x¢. with crystallit~e l v s o z v m e as s t a n d a r d . ('tw-.matagraphy K e t o acids were c o n v e r t e d into their 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e s• a n d s e p a r a t e d b y ;Lscending c h r o m a t o g r a p h y on W h a t m a n No. 4 paper. S o l v e n t s were b u t a n o l e t h a n o l - w a t e r (7 o : x o : 2 o ) Is a n d i s o p r o p a n o l - a m m o n i u m h y d r o x i d e - w a t e r (xoo: 5: Io) 16. F o r co]our d e v e l o p m e n t of '-rots, dried p a p e r s were d i p p e d in z°./o N a O H in 900,,5 eth an o l. Markers were p r e p a r e d from p u r e glyoxylic, p y r u v i c , o x a l o a c e t i c a n d a - k e t o g l u t a r i c acids x*. Bie,ckiTR. Bio~hys. Aaa, 67 0963} ~ 6 - 2 ~
ISOC['FI~ATE
LY.'..%F. A N D
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Isocitrate lyase G l y o x y l i c acid f o r m a t i o n w a s verified h v p a p e r c h r o m a t o g r a p h y <)f t h e k e t o a c i d p r o d u c e d . R v v a l u e s of t h e d e r i v a t i v e f r o m the r e a c t i o n m i x t u r e a n d its eel,tar r e a c t i n n w i t h ethane)lie N a O H were t h e .~ame a~ tho.,~e o f a u t h e n t i c gl.voxy]l¢ a c i d 2,4-dini iroplwtlv l h y ( l r a z o n e . -,-t,^. ,,. ~ c c t r u m .,,"¢ +~ ..... •. . ~.,.,...,,,' . . .........,. ;F.:St.÷;sVi;cz:'.qhydrazone. . c o n f i r m c . l the i d e n t i t y o f t h e k e t o acid. I t .~howed a m a x i m u m at 4So[tilL in a q u e o u s alkali, while tile r.~tio o f t h e a b s o r b a n c i e s a t 49o m/x a n d 540 ntlt was 1.84. T h e s p e c t r a ot ttle p y r u v i c a n d a . k e t t ) g h t t a r i c a c i d derivativt2.-..show a ph~tcau b e t w e e n these w a v t : l e n g t h s , w i t h a r a t i o a p i ) r o x i m a t i n g unit3:~, while a u t h e n t i c g l y o x y l i c acid d i n i t r o ~ , h e n y l h v d r a z t , n e h a s a r a t i o o f t. o. T h e m:mi-sl'mcific a n a l y t i c a l nl~,thod o f ~|Z'FADI.~EN ANt) HO~#,°ESte ()ft'('r~ f u r t h e r e v i d e n c e for g l y o x y t i c acid f o r m a t i o n , as it is spte.cific f~r aldeilyde.s t~. T h e s p e c t r u m o f t h e d e r i v a t i v e f o r m e d f r o m a u t h e n t i c glv~)xylic acid wa~ t h e s a m e as t h a t f o r m e d f r o m t h e r e a c t i o n p r o d u c t , s h o w i n g a niaxi~num ~lt 520 in/t~. T h e r e v e r . ~ r c a c i i o n o f f ~ r m a t i o t l o f i.-xwitric acid I'r~m g l y o x v h c at'id ~tnd s u c c i n i c a c i d w a s r e a d i l y d e m a n ~ t r a t e d 2, t h o u g h c h r o m a t o g r , t p ] ~ y failed to d e t e c t s u c c i n i c a c i d a f t e r t h e i s o c i t r a t e l y a s e reaction, it s e e m s r e a s o n a b l e to c o n c l u d e t h a t suc.cinic acid w a s f o r m e d d u r i n g r e a t t i o n a n d f u r t h e r m e t a b o l i s e d b v tile u n p u r i f i e d extract. I.-mci~.rate l y a s c a c t i v i t y o f u n p u r i f i e d extract.~ was c o m p a r a b l e t o t h a t relx)rted p r e v i o u s l y for s i m i l a r e x t r a c t s o f a c e t a t e - I z r o w n A. v i n e l a n d i i a n d A. agile t a n d o f P s e u d o m c m a s a e , u g z n o s a 2. T h e hibIhcst a ( ' t i v i t v o b t a i n e d xva.~ r e #mole.- t~f g l y o x v l i c acid p r o d u c e d p e r m g of p r o t e i n lXJr h, t h o u g h a~:ii,'~t~, d e c l i n e d slig|ltl~, ~-ith s t o r a g e . A f t e r 6 m n n t h s a t - - z o ~ in o.0z 5 M p h ~ s p h a t o buffer ( p H 7 o ) ceil-free e ~ t r a c t s r e t a i n e d 75~/0 o f t h e i r initial a c t i v i t v . Sucro.~e-grown cells s h o w e d negligable isoc i t r a t e lya.~e a c t i v i t y . Actl,mtio~z ~.4th ¢ y s t e i n e a n d E D T A T h e a c t i v a t i o n o f A z o t o b a c t e r i s o c i t r a t e l y a s e b y a r a n g e o f cv.~teine or E D T A c o n c e n t r a t i o n s is sh(Jwn in Fig. x. C.wteine is less effective t h a n E D T A . tn~th in e
~ X ~ r - (tYrA
oct~0r/3r~ Fig. I. The effect of cvs*.einc and ED'r. ~, on i,.~>cit~ate lyase acti~'ity of .4. vi~..la.dii $ ~xtracts.
E ~ h Thulthcrg tul0t- contaz~ned (in i~molesJ3.o ml final volume) : Trts buff~ar {pH 7.f>) zoo; MgCI=, (): ~='~DTAor neL~traliscO ~:y~teine hvclroCltlorit!c. ~ intlk:~ttcd; N~-oL-l.cdxaitratc. i n nit|c-arm. ,o; plus Aze~tobacter extract, l.o rnl~_ ' Reaction ~wL~ for 23 rain at 3o" after 5 rnin preincobation. Biechim. ~i¢~.hv~ j- . ° Acla, 67 {1963} ~zO--z39
I. R. K R N N E . D Y . M. J. D I L W O R T H
230
t e r m ~ o f c o n c e n t r a t i o n r e q u i r e d , a n d in m a x i m u m a c t J , , i t y r e a c h e d , T h e r a p i d d e c r e a s e in lya-~e a c t i v i t y a b o v e 2 m M E D T A is a p p a r e n t l y d u e t o i n h i b i t i o n cart -~d b y c h e l a t i o n o f Mg ~ . T o e s t a b l i s h t h e c o n c e n t r a t i o n o f M g t+ r e q u i r e d to m a i n t a i n m a x i m u m e n z y m e a c t i v i t y in t h e p r e s e n c e o f o.6 7 m M E D T A ( v ' h i c h w a s , , m p l e for a c t i v a t i o n ) , a s a t u r a t i o n c u r v e for Mg ~÷ w'a-~ d e t e r m i n e d . Since i ~ c i t r a t e lya.~e a c t i v i t y w a s o n l y s l i g h t l y b e l o w m a x i m u m in t h e p r ~ e n c e o f eqlximolar E D T A a n d Mg z~, ~ufficicnt Mg a* in s u b s e q u e n t e n z y m e a . ~ a y s w a s e n s u r e d b y use o f a 3-fold excess. A s c a n he .seen f r o m Fig. 2, t h i s d i d n u t i n t e r f e r e with the activating influence of EDTA.
3I
41'
.$/'
o
4
~,
~er~o e 5 ~ 1 ~ tml
Fig. 2. Isocitrate lyasc activation by Mg t-. Thunberg tubes contained reagents described under l~tg÷ x, except: A. p~tt."landii extract. 0. 5 rag; EDTA. z/~rnotes: MgCit, total am indicated, in fit~i volume of 3.o nd, Reaction was for x.$ rain ~t 30 ° ~J,fter 5 rain preingubation.
A d d i t i o n nf c y s t e i n e t o e x t r a c t m a x i m a l l y a c t i v a t e d w~th E D T A d i d n o t r e s u l t in increa,~ed a c t i v i t y ; in f a c t , l o w e r a c t i v i t y w a s r e c o r d e d . A c t i v a t i o n b y E D T A w a s a p p a r e n t l y r a p i d , for t u b e s w e r e i n c u b a t e d ':'or o n l y 5 ntin b e f o r e r e a c t i o n w a s s t a r t e d . I n c u b a t i o n o f e x t r a c t v d t h E D T A in t h e p r e s e n c e o f ,Mg~÷ in T r i s b u f f e r for p e r i o d s u p t o 6 h b e f o r e s t a r t i n g r e a c t i o n d i d n o t r e s u l t in a n y s i g n i f i c a n t c h a n g e in a c t i v i t y . T h e p a t t e r n o f a c t i v a t i o n o f A z o t o b a c t e r isocitl~ate l y a s e d i d n o t c h a n g e w i t h t i m e , E D T A still b e i n g s u p e r i o r t o c y s t e i n e i n a c t i v a t i o n a f t e r 6 - m o n t h s ' s t o r a g e at - - 20% T h e m e c h a n i s m o f b o t h E D T A a n d c y s t e i n e a c t i v a t i o n w a s a p p a r e n t l y t h e ,~-ame. W h e n p r o t e i n c o n c e n t r a t i o n w a s d e c r e a s e d f r o m x.o r a g / 3 m i t o o . x 2 r a g / 3 m l a n d E D T A c o n c e n t r a t i o n r e d u c e d t o 0.067 raM, i s o e i t r a t e l y a s e a c t i v i t y wa_~ o n l y 7 4 % 'ff t h a t o b t a i n e d w i t h 0.(> 7 m M E D T A . T h i s i n d i c a t e s t h a t E D T A a c t i v a t i o n w a s d e p e n d e n t o n its final c o n c e n t r a t i o n in t h e a ~ s a y m e d i u m , a n d n o t on a s t o i c h i o m e t r i c r e l a t i o n s h i p x~'ith t h e a m o u n t o f p r o t e i n .
zffect oj p n T h e e f f e c t ot pVi on J s o c i t r a t e l y a s e a c t i v i t y is sho~na in Fig. 3- M a x i m u m •, ~ v : : y ~ c e u r r e d b e t w e e n p H 7.6 a n d 8.o in n.o66 M T r i s b u f f e r . T h i s c o r r e s p o n d s c l o ~ l y t o ~he o p t i m u m o f p H 7.7 o b s e r v e d w i t h a n e x t r a c t f r o m P s e u d o m o n a s im~igofo'ais. a/oeAim. B/o-phys. Aaa. 67 (t963) 2a6.--239
IS(K'ITI~'~TE I.VASE AND TRIOSEPHOSPI~A-Vt,; I)HHYDROGENASI.:
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.__.J
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Fig. 3- p H depertdence_ of i~;0eitrate lya~e. "l'htznbtrr~ tubes contained (in ~umoIc.~/.~.om! final volume}; TI~.s buffer. -'tin; .3,IgCll, (~; EDTA, 2; ,Na~-~L-tsocttrat~. -<~; t;lu~ .'l. t'i~::'!an,t~i ex.*-ract, t.tJ mg~ I~.eaction wa.~ for Io miu itt .to° aftor 5 rain preincuhatiozt.
Protein concentration
When sufficient EDTA or cy~teine was added to achieve maximum activity of isocitrate lyase, glyoxylic acid formation with varying levels of Azotobacter extract w a s a s s h o w n i n F i g . 4. T h e p l o t for t h e s e r i e s w i t h E D T A a c t i v a t i o n is q u i t e l i n e a r a n d , u n l i k e t h a t with cysteine, passes thr~mgh the origin on extrapolation. At low protein cnnccn-
0.1
0.2 0.3 O.,a 0_5 nng ~c~erio~ proteir.
0.6
Fig. 4- 1-e~itrate ly~qe a c t i v i t y with increasing ct,;~centration of Azotobacter extract. Thunlx.rg t u b ~ c n n t a i n e d {in t=moles]3.o ml final volume~ : Tris bttffcr (pff 7.8}, zoo; Ms~('lz, 6; N,~-~LLsocttr~te, in side-arm, i5; ~"9---(). E D T A z; H . cystcine hydro~hloridc, t 5, R~-action was c o n t i n u e d for =-z rain a t 3o" after 15 mid p r e i n c u h a t i o n with .4. a,iT:¢landii extract added as inrlic;tbe~I. t r a t i o n s , c y s t e i n c h a s o c c a s i o n a l l y p r o v e d s l i g h t l y s u l ~ , , i o r t o E D T A (see T a b l e I l L s u g g e s t i n g t h a t u n d e r t h e s e c o n d i t i o n : ~ it e x e r t s s o m e u n d e f i n e d a c t i v a t i o n . I n c o m pa_,ison t o t h e E D T , ~ e f f e c t a t h i g h e r p r o t e i n l e v e l s ( F i g . 4), s u c h a n e f f e c t a p p e a r s to be very slight. B~ocklm. Biop~ys. Aa~. 67 (t963) zz~>-z39
232
|÷ R. KENNEDY. M. J, DILWORTH
Glyovvlate reaction w i t h thiol.s T h e depre.-sion o f i s o ¢ i t r a t e lya.-a.~ a c t i v i t y x~-ith h i g h e r l e v e l s o f c y s t e i n e (Fig. l) w a s itnlikclv to be cau.,~ed b y i n h i b i t i o n c o n . , ~ q u e n t u p o n M g t-~ b i n d i n g , as o c c u r r e d w i t h E I ) T A , for c y , t e i n e ha~ o n l y low a f f i n i t y fc~r Mg 2+ (log K less t h a n 4) t~. I n a d d i t i o n , t h e R~wcr a c t i v i t y :~bserved d u r i n g a c t i v a t i o n w i t h E D T A p l ~ cy.,.teine. r a t h e r t h a n E D T A alone, s u , . ~ e s t e d ~ o m e i n t e r f e r e n c e b y cy~teine. R e a c t i o n b e t w e e n cvsteint- a n d g l y o x y l i c a c i d is n o w well s u b s t a n t i a t e d *~,*~, a n d w e I~ave also o b s e r v e d rapid reaction between glyoxylic acid and cysteine at neutral pH. G l y o x y l i c acid c o u l d not b e detec.tt~i a f t e r a 1.5 m M s o l u t i o n w a s i n c u b a t e d for 30 rain at 3 o" w i t h a x6 m M s o l u t i o n o f c y s t e i n e in 67 m M T r i s b ~ f f e r ( p H 7.8) w h e n th:~ ~tnaivticai m e t h o d o f FI~IEDEI~A-~.TM ~,NI~ I-[AL'GEN~-~ w a s u~ed ( w i t h o u t s o l v e n t e x t r a c t i o n ) . \~'ith t h e m e t h o d o f McFA[)D~':y At~D HOWES ~*, t h e r e c o v e r y o f g l y o x y l i c acid w i t h h e a t i n g t i m e in p h e n y i h y d r a z i n e h y d r o c h l o r i d e at p H ~ wa~ as s h o w n in Fig. 5- T h e c o n t r o l series a l s o s h o w n in Fig. 5 c o n t a i n e d n o c y s t e i n e .
o
5
~3
T i r r ~ o f tte Qtin~ (rain)
15
F;g. 5- (~l>cTx)~'i~ a,:td re~:overy from ryst~-inu complux with heating titan. C,|yoxylatc was incubated at 3°" f(~r 30 mia in soluuon containing (in ~anolcslO.o roll: Tri~ bufft-r (pH 7.8) xooo; EDTA, 4 ; cvst~mc hydrochlot3tte, loo; ~lyoxylic acid. 9. A control contained no ~yst~inc. Colour proctuccd ~va_~ as shOWl~ wh~rn o.Z'05 ml sarnple~ were analyf~ett with pheny~hydrazinc hydrochlo:idc ~* by heating for vafiab],, lime at 95"'- G ..'~, plus cysteine; O---O, control. G r e a t e s t col()ur d e v e h ) l ) m e n t wa~ o b t a i n e d w h e r e h e a t i n g w a s o m i t t e d a n d t h e s a m p l e i n c u b a t e d w i t h p h e n y l l w d r a z i n e h y d r o e h l o r i d e a t r o o m t e m p e r a t u r e for 2o rain. T h i s e m p h a s i z e s t h e n e e d fu, ~:a~eful : 4 t a n d a r d i s a t i o n o f h e a t i n g t i m e , or i t s e l ) r e p l e t e o m i s s i o n , in a ~ r e e m e n t w i t h t h e c o n c l u s i o n s o f KRAM "R gl~ al. ~7 for a m e t h o d u~ing e s s e n t i a l l y t h e s a m e p r i n c i p l e . T h o u g h it is o b v i o u s t h a t h e a t i n g r e l e a s e s g l y o x y l i ¢ a c i d f r o m its c o m p l e x w i t h c y s t e i n c , t h e r a t e o f d e c o m p o s i t i o n o f g ! y o x y l i e acid p h e n y l h y d r a z o n e t o y i e l d a p r o d u c t w h i c h is n o t o × i d i s e d t o t h e f o r m a z a n m a k e s t h i s m e t h o d i m p r a c t i c a l f l e for t h e d e t e r m i n a t i o n o f g l y o x y l i c a c i d after incubation with cysteine. P e r c e n t a g e loss o f x m.~! g l y o x y l i c a c i d i n c u b a t e d witlt t h e s a , . c r a n g e o f c y s t e i n c c o n c e n t r a t i o n s a s u s e d for a c t i v a t i o n o f ist~citrate l y a s e (Fig, x) i.~ s h o w n in T a b l e I, T h e s e r e s u l t s s u g g e s t t h a t c y s t e i n e , a t s u f f i c i e n t l y h i g h c o n c e n t r a t i o n s , is a.s c o m p l e t e a n a c t i v a t o r as E D T A , a n d t h a t d i s c r e p a n c i ~ b e t w e e n t h e t w o r ~ u l t f r o m i n t e r action between glyoxylic acid and cysteine. 13iochim. 13iophy~. A ¢tu, t>7 (:963) 2~(>-239
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C o r x d i t i o n s o f a s s a y : ~ m+xl g l y t ~ x y l i c a c i d w a ~ i n c u bat~:ti .,it 3'~ , v i t h c y s t , - i , , , ' f , r -':~ m~.n i n 2 0 0 m.M T r i s b u f f e r {pP: 7A/.); z n , M S l g t ' t ~ ; c ~ b 7 m.'Xt E I ) ' I + . k . li~,al ".+~)!+,.,r~ .j+.u. ml t'glyc,xyl,~te in o. 2 4 - m l ~• d t l I l l l h + ~ "~,'~.~'i a q : ; a v o d
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~.Vhen g l u t a t h i o n e was te~ted fi)r interaction with g l y o x y t i c a~id in the syst~,m.~ described above, n o interfering effect coulcl be found. G l y o x y l i c acid recovery with z , 4 - d i n i t r o p h e n y l h y d r a z i n e ~ or p h e n y l h y d r a z i n e '~- was the ~atne as that in ccmtrol m i x t u r e s to w h i c h no glutathiom: had been added. ( ; l u t a t h i o n e has been reported to react w i t h c a r b o h ; l s ~ ; however, a n y c o m p o u n d formed here m u s t h a v e been easily disbociable. A clivity with time S p e c t r o p h 0 t o m e t r i c ag~lx' h,cthud.~ fi,r i~,. itr.~:;- l y a . ~ d e p e n ~ a n t on tile fl;rmalion o f c o m p o u n d s o f g l y o x y l i c acid with semicarbazide (~i;~,~rhing at z52 mt~. see ref. 24), and with p h e n y l - h y d r a z i n e hydrcwhioridc (absorbing at 324 m/z, see ref. 25), s h o w short lag periods before significant changes in ahsorbancv occur, probably due to slow reaction betw~en g l y o x y l a t e and the reagentg. W i t h A z . t o b a c t e r e n z y m e , and with direct chemical assay of g l y o x y l i c acid formation, the time course of i~)citrate lyase reaction v'r~ as shown in Fig. 6. The line o f best fit, b y the m e t h o d of least-squares, pas_c.es almost directly through the origd n + 1 .~,[
g E
b G L~
S C
&, ~) R,ea~--tk>n t i m e
~
15
(mll~)
F i g . 6+ I A n e a r i t y o f i s c ~ i t r a t e I)'a.~e r e a c t a o n w t t h t i m e . ~ V a r b u r g fla-~ks c o n t a i n e d (in l, m o l e ~ / 3 - 0 m l final r o t , . :,e) r "l'ris b u f f e r ( p H 7-6) 2 ~ ; MgCI=+ 6 : E D T A . z : N a = - D t . - i s o c i t r a t c {.~idt~-arrlt). 3 o ; p l u s A. uinetandB o~tt.rat:t, o+7,q m g . R e a c t i o n w a s s t a ~ e c i afte= a'" , n i n p r c i n c u b a t i o n a t 3 0 ° and stopped at the times indicated.
Bio~hi,n. Biopliya. +4tta, 67 ( t q 6 3 ) 2 2 6 - 2 3 9
234
I. It. K E N N E l ) V , M. J . DILWORTH
The effect of stcbstrate concentration Th e s u h s t r a t e of P. i~digofera i~ocitrate ly~se ha_~ been r e p o r t e d as I)B(+)isocitrate ts, t h o u g h o t h e r workers 2,s cons i de red the l.B(+)-form as t h e act i ve isomer for i,~citrate lyase from o t h e r microbes. DL-Isocitric acid was e x a c t l y h a l f as a c t i v e as the single i~omer fi>r isocitrate lya.ge, indicating t h a t onl y one form was m e t a bolised and confirming t h a t t h e i n a c t i v e fiwm p r o d u c e d no inhibitionS, a*. In this p a p e r we h av e followed t he n o m e n c l a t u r e o f VtCKERV za. who r e c e n t l y suggested an u n e q u iv o cal system to resolve t he present confitsed position. T h e E n z y m e Comnfission has r e c o m m e n d e d t h a t isocitrate lyase (form erl y isocitritase) be k n o w n s y s t e m a t i c a l l y as L~-isocttrate g l y o x y l a t e lyase. This should be superseded b y t hreol)s-i~x:itrate glyox_vlate lyase to agree w i t h t h e n o m e n c l a t u r e o f VICKERY tB for t h e n a t u r a l i.-~citric acids. T h e reaction rate c u r v e for s a t u r a t i o n o f A . vinelandii isocitrate lyase w i t h thre~-~>=-isr~citrate is shown in Fig. 7. a nd c o n f o r m s with ;~ICHAELIS--}IENTE.N"
i azl
1C/~
"~'--e'------------e
rrlM I'nreo -C~,- is,o c i t r o t o
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Fig. 7- E v a l u a t t o n of K m for i s o c t t r a t e lyase. " l ' h u n b e r g t u b e s c o n t a i n e d {in # m o l e s / b . o md final v o l u m e ) : "rris b u f f e r ( p l l 7-b) 400; MgL'It, t 2 ; E D T A , 4 ; t h r e o - o = - i s o c i t r a t e (as N a = - o L - f o r m i n s i d e - t t r m ) , as i n d i c a t e d ; p l u s .4. vi~ela~dii extr~vct, o - ] 5 m g p r o t e i n . T u b e s w e r e l ~ r e i n e u b a t e d &t 3o ~ for xo rain a n d t h e r e a c t i o n s t o p p e d a t 7 rain b y a d d i t i o n o f o . 4 ml o f 8o°/0 t r i c h l o r o a c e t i ¢ a c i d . R e s u l t s s h o w n a r e s i n g l e a n a l y ~ e s o n d u p l i c a t e d l e v e l s ot" s u b s t r a ~ .
kinetics. A LIXEW~:Avr:R--BuRK=¢ d o u b l e reciprocal ph)t of t h e r a t e of g l y o x y l i c acid forrr, atic;n r:cr,~ur, t h e thrc~, :;o-isocitlaL~: c , , n c e n t r a t i o n was linear, a n d e x t r a pointed to a Km o f 8.6- zo--" M. This lies within t h e r a n g e o f Km values p r e v i o u s l y r e p o r t e d o f 4.5" xo-~ M (see rcf. 2) a n d 5" zo-s M (see ref. 28). Purified y e a s t e n z y m e required t . 2 . xo" s M "I.s( + ) - i s o c i t r a t e ' " for h a l f - m a x i m u m a c t i v i t y s. A r e p o r t t h a t isocitrate lyase from P. indigofera was i nhi bi t ed b y c o n c e n t r a t i o n s of "l)0( i-}-isocitrate" g r e a t e r t h a n 8 . x o - 4 M , or of DL-isocitrate of x . 6 - x o - a b l (.gee ref. I8). was not in a g r e e m e n t with the present results. Because it was at first s u s l ~ c t e d t h a t this i nhi bi t i on was t h e result o f thiol r e a c t i o n with g l y o x y l i c acid a t higher cxmcentratinns of isocitratc, a nd he n ce o f g l y o x y l a t e , r a t e s o f r e a c t i o n ~Sth e i t h e r cy s tein e or E D T A as a c t i v a t o r s were followed. At low p r o t e i n c o n c e n t r a t i o n (4o p4g/ml) w i t h low g)yoxylic acid p r o d u c t i o n , a c t i v a t i o n b y c y s t e i n e a n d E D T A wa_~ closely similar. As shown in T a b l e II, no inhibition was o b s e r v e d at c o n e e n t r a Bioc~im B i o ~ s . Acta, 67 (t~3) 2=6--z39
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Conditions of as,gay were :ts d.r~ribed in Fig. 7 ~-xccpt: .-I ~,inela~ldti extract, 0.-- 4 mg vr~,teia; reaction was for ~z rain m 6.o-m! t-:rml volume. G;;mr'¢l:'g :~r ~:~ [.,~me,! (~.m,cdtJ ~~ * ' )
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t i o n s o f u L - i c ~ 3 c i t r a t e u p t o 8 . s o a M. C l e a r l y +'.,r~ i ; i h i b ; t l o n o b s e r v e d In is n o t d u e t o a r e a c t i o n o f t h i s t y p e , e s p e c i M l y ~ g h t t a t h i v n e w a~ u s e d f o r a c t i v a t i o n . I t t h e r e f o r e a p p e a r s t h a t s u b s t r a t e i n h i b i t i o n is n o t a g e n e r a l p r o p e r t y o f i s o c i t , ' a t e lya.~e.
T r i o s e p h o ~ p h a t e dehydroge~rase T h e p r e s e n c e o f t h i s e n z y m e i n A . ~'inda, d i i h a~ b e e n i n f e r r e x t f r o m i n d i r e c t e v i d e n c e b y M o R ' r E ~ S O N , H.~,HtLTO.~ A.~r> WtL.~O.', m. A l t h o u g h d i r e c t r e d u c t i o n o f N A D d i d n o t o c c u r in a n e x t r a c t w i t h a d d e d f r u c t o s e d i p h o . ~ p h a t e a n d a l d o l a s e
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ltiochi,n. Biophys. .4cJa, 67 (I963) z26--z39
236
I. R. KENNEDY, M. J. DILSVORTH
(IsC 4. I.2.7), N A D H 2 o x i d a t i o n could be d e m o n s t r a t e d in such systems. S u b s t a n t i a l N A I ) r e d u c t i o n has been a c h i e v e d w i t h p a r t i a l l y purified enz3m~e from A. vinelandii. As shown in Fig. 8, x mbl E D T A was slightly b e t t e r for a c t i v a t i o n of N A D r e d u c t i o n b y g l y c e r a l d e h y d e 3 - p h o s p h a t e and A z o t o b a c t e r e x t r a c t t h a n 3-5 mM cysteine. Sl:.(;,xL ,XXD BO~'ER r h a d n o t e d a similar SUl~riority o t" E D T A over cysteine for a c t i v a t i o n of t r i o s e p h o s p h a t e d e h y d r o g e n a s e from muscle. P y r o p h o s p h a t e g a v e g r e a t e r a c t i v i t y than did Tris of the s am e p H a n d m o l a r i t y (22.5 raM] a n d m a x i m u m a c t i v i t y was o b t a i n e d at p H 9.o. Unlike t h e p r e p a r a t i t m o f St-:t;Al. AXlJ BOYEU 7, Tris buffer did llot allow a l o n g : r pc.clod o f lineal a b s o r b a n e y change. N A D P was not r e d u c e d in t h e s ys t e m . Th e g l y c e r a i d e h y d e 3 - p h o s p h a t e c o n c e n t r a t i t m for h a l f - m a x i m u m react i on velocity wa.~ e~timated er at 5.3" I o- s M a t p H 9.~) and 3o ~. An a t t e m p t was m a d e to a c t i v a t e the e n z y m e with cysteine, a n d t hen to r e m o v e the excess b y dlalv~i~. . _. 2 ml of enzyme. (4.6 m g of protein) were t r e a t e d w i t h 0.0 5 ml " f ~ 2 ~t cvsteine h y d r o c h t o r i d e a t p H 7.z for z h at o ° in a T h u n b e r g t u b e u n d e r nitrogen. T h e e n z y m e was q u i c k l y t r a n s f e r r e d to dialysis t u b i n g ( E D T A washed) and dialysed x~-ith stirring against x 1 of deionised w a t e r in an a n a e r o b i c F i l m e r j ar filled xwith nitrogen and c o n t a i n i , g an o x y g e n a b s o r b e n t . A f t e r x8 h dialysis, tlie e n z y m e was r e m o v e d a nd a,~sayed withol, t eyste.ine. No increase in a c t i v i t y a b o v e t h e u n t r e a t e d e n z y m e could be d e m o n s t r a t e d , a l t h o u g h t h e dialy.',;cwl e n z y m e coul d be r e a c t i v a t e d b y c.x,steine to an a c t i v i t y identical with t h a t f o u n d a f t e r t he initial cvsteine activ atio n. This result indicates t h a t t h e r e is a r e q u i r e m e n t for e o m p l e x i n g r e a g e n t in t h e m e d i u m during e n z y m e action, and poi nt s to the possibility t h a t the a c t i v a t i o n was c o n c e r n e d with a p r o t e c t i v e effect o f cy~teine ag,tin~t o t h e r c o m p o n e n t s o f t h e reaction m i x t u r e . DI.qCt;.q.qION In a g r e e m e n t with previous findings (see KOI~NI~I.'.HG~ for review), i.~mitrate lyase has again p r o v e d a d a p t i v e to g r o w t h on acetate. During g r o w t h on .sucr.~e. which tl()es not require net synthesis of d i c a r b o x v l i c acid.;, no isocitrate lya-.;e a c t i v i t y was .'letccted. T h e finding t h a t E D T A is an efficient a c t i v a t o r ,)f i s . c i t r a t e lyase assumes Slx~cial signilicance because of the serious i n t e r f e r e n c e in ashtray b y cysteine. Reactions of t h e t y p e occurring b e t w e e n g l y o x y l i c acid and cysteine are now well docum e n t e d . T h e ease of reactio:~ b e t w e e n c y s t e i n e a n d k e t o n e s at n e u t r a l p H a n d r~×)m t e m p e r a t u r e h as been noted'~L G l y o x y l i c acid a n d cysteine yield a s u b s t a n c e which o n l y r eacts wit|. p h e n y l h y d r a z i n e at m o °, a n d form s a thiazolidine qui t e different f r o m t h e n o r m a l h y d r a z o n e f o r m e d in t he absence of c y s t e i n e % t h u s explaining inc o m p l e t e recoveries of g l y o x y l a t e . T h e seriou~sne~s of the react i on b e t w e e n g l y o x y l i c acid an d cysteine has r e c e n t l y been r e l a t e d t o t h e assay of i s o c i t r a t e lyasetL T h e f o r m a t i o n o f a c y s t e i n e - g l y o x y l a t e c o m p l e x m a y be c o n s i d e r e d t o p r o d u c e t w o results: (a) an a p p a r e n t lowering o f a c t i v i t y d e p e n d e n t on r e c o v e r y of g l y o x y l a t e , w h i c h will be a f u n c t i o n of t h e time ~f e n z y m e assay used, and t h e degree of h y d r o lysis in t h e a n a l y t i c a l m e t h o d (a s a t i s f a c t o r y m e t h o d for h y d r o l y s i s of t h e c o m p l e x sb-ould o b v i a t e difficultie.~ ee.u'~--d b y its f o r m a t i o n t t ) ; ( b ) a lowering of a c t i v i t y d-~pending on the e x t e n t o f r e m o v a l o f cysteil~e b y g l y o x y l a t e . D uri ng t h e course o f B i o c A i m . B~opky.~. Ac/a. 67 (t9~$) 2 z 6 - 2 3 9
ISOCITRA'rE
].VASE
AND
TRIOSEPHO-'gPi!AI'E
DEH'~'DROGENASE
237
e n ' ~ y m i c r e a c t i o n , a c o n t i n u o u s l y d e c r e a s i n g cy.~teine c o n c e n t r a t i o n t m a y he e x p e c t e d t o m a k e t h e d e t e r m i n a t i o n o f a b s o l u t e a c t i v i t y ditlicult. I t is t h e r e f o r e i n a d v i s a b l e t~ use cy.steine for i s o c i t r a t e l v a s e a c t i v a t i o n . G l u t a t h i o n e is a p p a r e n t l y a s u i t a b l e s u b s t i t u t e , b u t E D T A h ~ t h e a d v a n t a g e t~t" ...tAt n ~ t b e i n g a ~ t o x i d i s a b i e , a n d .N.tL~...... thereful~, aihJw a e r o b i c a s s a y , f i~ocitrate lyase. T h e i m p l i c i t a s s u m p t i o n in cv~tein,: a n d g l u t a t h i o n e a c t i v a t i o n o f i ~ o c i t r a t e lya~e h a s l~een a specific effect o f t h e i r -.%H g r o u p cm t h e e n z y m e m~flec~]Ie. [3} c t m t r a s t , E D ' F A d o e s n o t pos.~c~ .inch a g r o u p . It w o u l d t h e r e f o r e appe~tr t h a t exp l a n a t i o n s i n v o l v i n g cy.~teine in r e d u c t i o n of e n z y m i c - S H grlmups a r e improl~al~le in c o m p a r i s o n w i t h o n e i n v o l v i n g b i n d i n g o f h c a x ' y - n t e t a l ions f r o m t h e etnzyme. T h e n o n - a d d i t i v i t y - f t h e c y s t e i n e a n d E I ) T A effe~t~ is s u p i , o r t i n g e v i d e n c e t't~r t h e similarity of their action. S t i m t t l a t i o n b y m e t a l - c h e l a t i n g s u b s t a n c ( ~ h~t~ b e e n sho~vn f,~r a widt: r a n g e or" enzyme_~. D l ; P a s e fEC 3.,q.2.i) is activatetq h'~, 2 , 2 ' - d i p y o d y l a2, K ~ - a ( : t i v a t e d a l d e h y d e dehy(lrt~gena~e (EC 1.2. I . 5 | l)v t h i ~ l ~ , h i s t i d i n e a n d EI)T:X ~. a n d ~'isa c o r d t i c decarboxyla_,~, (EC 4 . I . i . 6 ) b y c:.-.~t~,ino, E D T A a n d 8-hydr,~xx'quinoli~te '~. C h e l a t i o n o f i n h i b i t o r y m e t a l s h a s b e e n s u g g e s t e d as t h e m e c h a n i s m o f a c t i v a t i o n b y E D T A o f m u s c l e t r i o s e p h o s p h a t e dehvdragc~a_¢,e (EC 1.2.~.[2) ~. heart-mu.~:l,o x i d a s e p , ' e p a r a t i o n s as a n d p h o s p h o g h ~ c o r n u t a . c e (I"C 2.7.5. I ) ~ . a L P u r i f i e d y e a s t i . ~ c i t r a t e l y a s e ,.'~ i n h i b i t e d b y low c o n c e n t r a t i o n s o f Cu ~-'. ( ' d ~a n d Zniv0 w h i l e s u b s t a n t i a l i n h i b i t i o n o f isocntra:e l.x'ase t m c u r r e d in lo -r .X[ p c h l o r o m e r c u r i b e n z o a t e , a n d this i n h i b i t i o n w a s 8o';.~ rever.~ed b y ex~:e~s cvstei~te ~. Tiffs p o i n t s to t h e pL'u>ence o f an e s s e n t i a l - S H g r o u p in t h e y e a s t e n z y m e , a n d it is t h e r e f o r e l i k e l y t h a t a c t i v a t i o n . f A z o t o b a c t e r ettz)'rne b y c y s t e i n e a n d E D T A is t h e r e s u l t o f c h e l a t i o n o f i n h i b i t o r y h e a v y m e t a l s b o u n d t,~ it, p e r h a p s to a n .%I1 g r o u p . T h e p o ~ i b i l i t y o f o t h e r m e t a l - b i n d i n g s i t e s r e m a i n s : itt.s'rv,'~N az p r e s e n t s e v i d e n c e t h a t Z n =" i n h i b i t i o n o f phosphoglu~~m~utiL~e is n o t d u e to t h e f o r m a t i o n o f a .-ilercaptide a n d s u g g e s t s a l t e r n a t i v e b i n d i n g s i t e . ~ - - a m i n o , intidazoly[, t'nrbt~xvI and phosphate. T h e c o n c e n t r a t i o n or" c y ~ t e i n e recluir~"l t o a c t i v a t e i s o c i t r a t e lya~e in A z o t ~ b a c t e r e x t r a c t s is c o n d i d e r a b l y g r e a t e r t h a n t h a t o f E D T A . Allowing G>r i n t e r a c t i o n o f g l y o x y l i c a c i d w i t h c y s t e i n e , t h e con~centrath~II t~f t h e thio] r e q u i r e d i~ a t l e a s t 3 raM, w h i l e w i t h E D T A , o.z'.: m M pr~Xucc:~ a l m o s t m a x i m u m a c t i v i t y . I f a m e t a l ion is re_~ixmsible for t h e inactivt" fi>rrn o f i s o c i t r a t e lyase, it appeztn's t h a t cy~t,-ine is less efficient in b i n d i n g t h i s m e t a l . F r o m t h e s t a b i l i t y c o n s t a n t s for c o m p l e x e ~ b e t ~ ' e e n v a r i o u s m e t a l ion~ a n d e i t h e r c y s t e i n e or E D T A t~, c a l c u l a t i o n s w e r e m a d e o f t h e r a t i o o f free to b o u n d m e t a l for a r a n g e o f ions, m a k i n g a l l o w a n c e for f o r m a t i o n o f z : 2 c h e l a t e s betw<~n s o m e m e t a l s a n d c y s t e i n e . F o r o . I 6 m M l i g a n d {the E D T A c o n c e n t r a t i o n g i v i n g m a x i m u m a c t i v i t y , see Fig. z) a n d a contstant m e t a l - i e m level, t h e equilibrium c o n c e n t r a t i o n o f free Co -~~, Fe ~', .~i a-, PU ~ , Z n a~. Mg ~" atnd Mn ~+ w o u l d b e g r e a t e r w i t h c_vsteine tlnan ~ ' i t h F, D T A , while t'.,te rever~e w o u l d b e t r u e fi~r F e ~- a n d H g * ' . I t is therefi~re u n l i k e l y t h a t t h e l a t t e r ioais a r e r e s p o n s i b l e f~>r t h e i n a c t i v e f o r m o f i s o c i t r a t e l y a s e O f t h e f o r m e r , Mg 2¢ , M n 2", Fe"" anti ( ' o z ~ h a v e b e e n s h o w n t o a c t i v a t e i s o c i t r a t e lyaseS, ~.m, m a k i n g it u n l i k e l y t h a t t h e y a r e c o n c e r n e d in its i n a c t i v a t i o n . A l t h o u g h a s t a b i l i t y c : m s t a n t for t h e Cd~'-~=ysteine corr~p!ex i~ n o t a v a i l a b l e , it is /3i,J~ h~m. Bi,pA~'s. A*'ta. 67 (:9~,3} ~2f~--~39
I. R. K E N N E D Y ,
z38
M. J . I)ILXVORTH
p r o b a b l y similar t o t h a t fi.~r t he CdZ+--glutathione cnrnpiex, in which a lower proportion o f m e t a l is b o u n d thm~ in the C d ~ + - E D T A complex. I f these idealised chemical d a t a are applicable to the s y s t e m used for isocitrate ly.~e assay, then, of the cases available for c o m p a r i ~ n , the ions m o s t likely to cau.~e i n a c t i v a t i o n are Zn*", Ni a ' , I+b~+ a n d Cd +'. It is ix)ssible t h a t Cu ~+ is also i n v o h ' e d , A l t h o u g h d e p a r t u r e s from i de a l i t y are o b v i o u s (in t e r m s of p H , t e m p e r a t u r e , ionic s t r e n g t h an d o t h e r ions), the differences in ratios of free to l x m n d m e t a l for E D T A and cysteine are so large t h a t some assessment of t h e relative a d v a n t a g e s of t h e e c<>mi~mnds is ~ i b l e . A v a r i a t i o n in rt~Imnse can be e x p e c t e d duri ng a c t i v a t i o n b y different c h e l a t i n g :.ubstances, the a c t i v a t i n g efficiency o f w hi ch will be a f u n c t i o n of t h e t y p e a n d quantitit-'s o f th e c o n t a m i n a n t s present, a n d not necessarily" a c o n s t a n t p r o p e r t y of the en zy me. It is t h e r e f o r e possible t h a t t h e ~ u p e t i - r i t y of E D T A over c y s t e i n e will l l O t alway s be observed, a l t h o u g h in the m a j o r i t y of caa~s considered above, E D T A ~em<>val ,)f metals .~houh] be m o r e c o m p l e t e . ACK
Nowl.]:. Df;EM ENTS
Gri,*eful acknowlcxlgment is m a d e t o Dr. C. A, PARKER for his c o n t i n u o u s e n c o u r a g e ntent a n d to Professor E. J. U.X'DEmWOOD, D i r e c t o r of t h e I n s t i t u t e , for his s u p p o r t . A c k n o w l e d g e m e n t for financial assistance is m a d e to t h e C o m m o n w e a l t h Scientific a n d I n d u s t r i a l Research O r g a n i z a t i o n for s t u d e n t s h i p s , a n d to t h e U n i v e r s i t y o f W e s t er n Au.~tralia for re qearcl, grants. REFERENCES ~r~. A. S.MtTH A~D I. C. GL'.N'S^LL'S, .%'adUrt, x75 ( ] 9 5 5 ) 774-
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