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Inhibitor, isotopic and kinetic studies on hydrogen dehydrogenase
BIOCHIMLCA I'~T BIOPI'{YSICA ACTA
~9
neA z2x59
INHIBITOR, ISOTOIqC AND KINETIC STUDIES ¢)N H Y D R O G E N D E H Y D R C b G E N A S E D. ][. i$ONK • Deparlm¢):t ~ f ,a,Z icrc'~.i,plo;:y, $'¢*l¢ L; )r it', r.set),, Neu, H a v e n , Co))n. ( U..g..-I .)
( l t e c e i v e d A u g u S t z 7 t h , nqt,.,.)
SUMMA L,,L Y T h e oxidized form of h y d r o g e n dehydrogena_se (,f H vdrogt'~tzomo~:as rJrJdamtii was in* hibited b y BAL. cyanide, dithianite, sulfide. ~uifite a n d thioglycolate. Reduction of the d e h y d r o g e n a s e with DPN H or I'I~ resulted in a dindnished effect of the inhibitors, w i t h the exception o f p-chiorome=curibenzoate which inhibited only the reduced e n z y m e . I n f o r m a t i o n from kinetic analysis and inhibit(~r studies indicates t h a t the e n z y m e is reduced with H , to give a proton and t h a t this i~ followed by the oxidation o f t h e reduced e n z y m e w i t h D P N to gi;'e DPN H. The enzyme catalyzes an exchange between H~ a n d H T O a n d also between HTO a n d D P N H , Analysis of DPN enzymically reduced with T a shows t h a t 1" is in the//-position (,f DPN H. The e n z y m e actic~n h a s a lag phase which (~an be eliminated b y preincubation with I-I~ or D P N H . llgTICt)[~UCTION
T h e m e c h a n i s m o f action of h y d r o g e n a c t i v a t i n g en/.yme~ has been investigated from the viewpoint of exchange reactions u,2 the metal comlx)nenO,' and a possible flavin comportentr-L Th," hydrogenase o f Desulfovibrio destdfi~rlcav.s has been e x t e n s i v e l y investigated :.4.~' a n d RaKI.Is ,x~i) R r ~ E x ~ r t 6 ~ have o b t a i n e d a highly purified preparation using the exchange reaction betwL~en deut,:rium oxide am] h y d r o g e n as the a s s a y system. The hydrogenase of D. desulfi,Hcans can aIso re~luce benzyl viologen, m e t h y l e n e blue a n d liberate h y d r v g e n fn)m reduced m e t h y l vioIogen*, a. Spectrographic analysis o f this protein showed iro,~ to be the major metal component. The exchange reaction indicated t h a t the hydrogen;~se reaction can occur as a heter(flytic proce.~ resulting in the formation of an enzyme h y d r i d e and a proton ~. H y d r o g e n d e h v d r o g e n a ~ of Hydrogenomonas ruhlandii provides a more amenable system for the s t u d y o f the activation o f hydrogen and in this report a t t e n t i o n has been focus.ged on inhibitors which cat) react with disulfi~le groups, the u~e of i-~otop,~.~ to evaluate the exchange reaction.~ and a kinectic analysis of the h y d r o g e n d e h y d r o g e n a ~ reaction. A b b r e v i a t i o n : I-~CMLL p-chluromecuril~.'nzo~Lte• I~reoent a d d r e s s . D e p a r t m e n t o f B o t a n y . Q m ' o n M a r y College. U n i v e r a i t y o f L o n d o n , L o n d o n E . z ( G r e a t lJritain)÷ t4toch;m, i d i o p h y s . . 4 c l a . 67 (1063) 5rig-.508
5fjO
D.H.
BONE
MATERIALS AND METHODS
T h e f.)llowing m a t e r i a l s w e r e c o m m e r c i a l p r e r ~ a r a t i o n s : p i g - h e a r t m a l i c d , : h y d r o g e n a s e {C F. B o e h r m g e r a n d .qohne, M a n n h e i m , G e r m a n y ) , l i v e r - g l u t a m i c d e h y d r o g e n a ~ (Sigma C h e m i c a l C o m p a n y ' ) , D P N a n a l o g s ( P a b s t L a b o r a t o r i e s ) . I l y d r o g e u d e h y d r o g e n a s e u s e d w a s e n z y m e B a n d a s s a y e d as d e s c r i b e d in t h e a c c o m p a n y i n g patx:ra°. K i n e t i c m e a s u r e m e n t s w e r e m a d e using o . x M T r i s - H ( ' l buffer ( p H 7-8) m t u r a t e d w i t h H~ a t 25 °. T h e s o l u b i l i t y o f H t in Trig b u f f e r is a.~sumed to be t h e s a m e as in w a t e r . T h e t e c h n i q u e for t h e m e a . ~ u r e m e n t ~f r e d u c e d a n a l o g s of I ) P N is t a k e n f r o m t h e P a b s t L a b o r a t o r i e s C i r c u l a r O R - x S . S a m p l e s o f t r i t i a t e d m a t e r i a l w e r e p i p e t t e d o n t o p l a n c h e t t ~ a n d t h e n d r i e d d o w n E a c h .~ample w a s t a k r n u p in 0.2. m l o f w a t e r a n d e v a p o r a t e d t o d .r3,ne~g 5 times. R a d i o a c t i v i t y w a s t h e n m e a ~ x r e d u s i n g a windowle~q gag flow c o u n t e r . RESULTS
I nhibdor.s T a b l e I s h o w s t h e effect o f i n h t b i t o r s on h y d r o g e n d c h y d r o g e n a s e . T h e i n h i b i t o r s listed are r e a g o n t s w h i c h split dis ."tale b o n d s n a n d a]so can, in t h e c a s e o f c y a n i d e a n d s u t h d e r e a c t w i t h a m e . t a P a. T h e r e a c t i o n o f c y a n i d e a n d .~,ulfide w i t h t h e o x i d i z e d e n z y m e i~ a slow reactior, a n d t h e m a x i m u m i n h i b i t i o n is a t t a i n e d o n l y a f t e r a 5-rain i n c u b a t i o n p e r i o d w h e r e a s sulfite r e a c t s ~ n s t a n t a n e o u s l y w i t h t h e e n z . v m e a n d TABI.E F2FF/~I2T O F I N I I I B I T O R S
1
ON MYDRO4",RN DE~VDROGENASF.
ACTIVITY
l'rc~ncubAt~d solution.~ contninexl Ip'drogen dehydtoguna~ t~ufficient enzyme to give an t~bsorba u c y c h g n g e a t 34 ° m p o f o . z t r n i n in t h e f i n a l v o l u m e ) , adtt| 2 o / ~ r n o l e s T r i g | [ C l b u f f e r { p H 7.8}. For DPNII preincubation, the m,hnion contained enzyme. 2o/~rnoles "l'ns-succinate (pH 6.9) a n d o o b / c r n o l e L ) P N H . I n h y d r ~ g t . n p r e i n c u b a t i o n e x p e r i m e n t s , h y d r o g e n ,a a s Fm,sstx| t h r o u g h the solution. Prclncubatlon w a ~ .5 xnin a t z 5 ~. v o l u m c o f s o l u t i o n t,.2 m l a n t i c o l ~ c e n t r a t i o n o f i n h i b i t o r s a s in
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K C N ( p H 7.8) K ( ' K (1,H 7.8) + KCIK { p H 6 . 9 ) K C N { p H 0.9~ + K C N (pH 7.8) -M N a t S (I', I 7.8) N a t S ( p { 7.8j M N a t S ( p H 6.9) M N a t S ( p H b-9} M N a t S ( p H 7.8}
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5 " ~o-~ M N g t . q ( p H 7.8") 1o +a M N a t S O a ( p H 7.8) IO -a M N a t . S O , ( p H 7.8) + H s 5 " ] ° ' s M N,'htSsO a 5" t o - i M N a z S a O , 4- | ' I s 3"lo*M BAL 3 1o-* M B A L ~. I t e 5 " xo-~ M T h i o g l y c o d a t e 5 • t. i M T r . i o g l y c o l l a t e 4- H s 7"z, -M t'CMU 7"1o-¢ M I~CMB ¢ l| t
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Bio~him.
Fl:optD's..-Iota.
67
(1963) 5~9-598
FI~'I)ROGEN DEHY!)R|Jt;EN:kSE
59~
t h e r e a c t i o n is ~ompLet,.. in 30 ~ec. A t t e m p t s t(~ r e v e r s e ttJa_~ i n h i b i t i o n s o f N e z 5 ~_ad K C N b y (a) d i l u t i o n t h i r t y - f o l d witl, "l'ris buffer (b) r e m , , v a l o f i n h i b i t o r b y 5-'pha~lex G - z 5 gel raider conditirDns wtfi~-h efftn'Zivelv r e m o v e sulfide ( T a b l e 1) (cl dialyai.~ a g a i n s t t o o v o l u m e ~ o f o.05 M pota.~sium phosphatt~ ( p i t 7.,~). o.,~oz M E D T A o.o~Jqr/M MnCI. for z 4 h (d) treatmer,-: w i t h t~.ooz 31 ZnSO~ (e) additi,~,n o f 0.03 3I i x ) l a s s | u r n s a l t o f E I ) T A , w e r e not ~-ffcctix-~. T h e o n l y c o n d i t i o n w h i c h varti~:liy rev e r s e d t h e i n h i b i t i ( m o f sulfide wa.~ t h e d i l u t i o n t,t' t h e p t e i n c u b a 0 u t a m i x t u r ( . ,v'~eh a s o l u t i o n ()f (~%5(_; ( T a b l e I}. A i ) P N - s u l f i d e e n z y m e c o m p l e x h a s I-,~.~.,~ rep..~rt,~d a s t h e m e c h a n i s m ,~t"i n h i b i t i o n o f !:.,~th: ( l e h y d r o g e n a , e ~ a ; b u t in t h e case ~f h y d r o g e n d e h y d r t ) g e n a s e D P N h a d r.~ effect on tlk- inhibiti,,n ~f t h e , , ~ z y m e b y 2 - z o ~ M NaaS. F u r t h e r , D P N w a s f ~ u n d to h a v e nc) effect ~,~ ,tnv ()f t h e inhibitor~ e x c e p t in t h o s e k n o w n cu_ses when" ! ) P N r e a c t s d i r e c t l y w i t h the irthil)it~)t÷ T h e r e a g c n t ~ w e r a all n-,c,l~- ~fft-~tiv,: wht.l~ tht- i n h i b i t ~ r wa~ preie~c,~bated ~'~th t h e e n z y m e a n d the teacti~)n ~ t a r t c d b y a d d i n g I 2 : a n 0 I ) P N t h a n w h e n t h e e n z y m e w a s first r e d u c e d b y I] z or D P N H a n d t h e inhibilt~r t h e n a d d e d T h e e x t e n t tn ,vhich t h e p r e t r e a t m a n t a f f e c t e d t h e inhibitio.~ w..s ( l e p e n d e ~ t ~,n t h e i n h i b i t o r ( T a b l e I). V a r i a b l e r~-~;ult.~ fo- cynic|de i n h i b i t i o n h a v e i~.t.n r e p o r t e d for hydr~,~enase.~ f r o m a n a e r o b i c ba~teria~-'~.e.a. ~. T h e at~thor h~t~, t2~und r ~ a t ~i i~ e.~ct~tial to ~ t a n d a r d ize c o n d i t i o ; : s ft~r all i n h i b i t o r studie., a n d t h a t ~l_~t.~-ial c a r e be g i v e n to the. o x i d i z e d r e d u c e d s t a t e o f t h e e n z y ' m e , if e a r | a i s l e rcsult.~ are to he e l i m i n a t e d . I t w a s p o s s i b l e t o c a r r y ~)ut a r e d u c t i o n o f hyclr~gen d e h y d r o g e n a t e w i t h a s m a l l a l n o u n t o f H~ (0.3/~mole) to g i v e a n e n z y m e w h i c h is i n h i b i t e d 4-F'~'~, b y 5 " x o ~ M Na~S. W h e n t h e e n z y m e w a s tht:n o x i d i z e d w i t h 3 ~um~)le~ o f I ) P N a n d t h e nu~:let)tides f e i n t ) r e d w i t h S e p h a d e x G - 2 5 gel, a n , : n z y m e w a s o b t a i n t . d w h i c h is c o m p l e t e l y inh i b i t e d b y sulfide. P C M B i n h i b i t i g n v~tries in the r,:verse m a n m : r to t h a t o f ti~e o t h e r i n h i b i t o r s . t h e r e d u c e d enzxa~ne b e i n g m o r e s e n s i t i v e to P('.'~ B ( T a b l e I). A .~imilar effect w a s n o t i c e d w i t h glutathi~H~e reducta.,~, which bec'~me~ ~en~itivc t() ~ulth3"dryl r t . a g e n t s w h e n r e d u c e d w i t h T P N II o r D P N H ~. A n , ) t h e r inhibit,~r .~tu(t~t.d was xo ~ 5I a r s e n i t e w h i c h i n h i b i t e d t h e e n z y m e ~e"i~ w h e t h e r t h e et~z)'nl~, xwle, ~xidizcd or r e d u c e 0 . T h e effects o f v a r y i n g t h e c o n c e n t r a t i o n t)f N a t l . u r e a a n d T r i s - H C l bufft~r (pI-t 7.Y) w e r e t r i e d in a r s e n i t e i n h i b i t i o n e x p e r i m e n t s using e n z v m t . s A a n d pl~0 a n d enzvn~e B p r e p a r e d in t h e a b . ~ n c e o f m a n g a n e s e . I n n~) case w a s t h e i n h i b i t o r y effect ()f a r s e n i t e |not'eased. O x y g e n w a s f o u n d ~ o t to inhibit t h e e n z y m e a n d nn precaution_~ w e r e f o u n d n e c e . ~ r y tt~ e n s u r e t h e a b e - , ~ o f o x y g e n d u r i n g t h e purilicatic)n pr(~cedure t°. A s sulfide m a y fi)rrn a s t a b l e c o m p o u n d w i t h t h e e n z y m e , the e n z y m e (~oo ~g) w a s i n c u b a t e d w i t h NaaaaS ( o . r / a m o h : , 2 - x o ~ eount.~;mit~) for zo rain a t r o o m t e m p e r a t u r e . T h e sulfide a n d e n z y m e w e r e s e p a r a t e d b~" t h e a d d i t i o n o f sufficient .~aturated (NH~).,qO, st)lution t~ b r i n g the s a t u r a t i ~ m to 6o0~. T h e p r e c i p i t a t e d p r o t e i n w a s w a s h e d t w i c e w i t h 6 o ° ~ saturat,~d (N[I.,)~S() a a n d its r a d i o a c t i v i t y t h e n m e a s u r e d . N o t-ad:'oactivity w a s a s s o c i a t e d w i t h t h e p r o t e i n .
tginelics of inhibited enzyme Tile e f f e c t s o f t h e inhibitt~rs c y a n i d e , sulfide :rod ~ulfite w e r e i n v e s t i g a t e d b y m e a s u r i n g t h e r:Lte-n o f r e a c t i o n in the p r e ~ m c e ()f a c o n s t a n t a m o u n t o f e n z y m e , i n h i b i t o r a n d e x c e , ~ .~uh~trate, whilst v a r y i n g t h e c o n c e n t r a t i o n o f t h e o t h e r s u b Bi.ch~m l~ophys..4ct~. /J7 ('(c)63) 589-598
D . H . nONE
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Fig. ]. Effect of cyanide, sulfide and sulfite on hydrogen dehydrogenase. 0.0 4 unit of oxidized enzyme preincubat~_~l ~ in Tahle I with ]o -i M KCN (Curve a). to -a M NaaSO. |Curve 3) and z-5 ÷xo -¢ M Na,S (Curve t)- Reaction started by adding varying amounts of D P N and H a saturated buffer.
s t r a t e . T h e L i n e w e a v e r - B u r k p l o t w a s u s e d t o a n a l y z e t h e r e s u l t s (Fig. t). All t h r e e i n h i b i t o r s were c o m p e t i t i v e w i t h D P N a n d p a r t i a l l y n o n - c o m p e t i t i v e w i t h Ha.
Kinetics of red,ction of D P N ~_ hydrogen A lag p h a s e w,~Ls . h ~ e r v e d w h e n t h e e n z y n l e w a s a s s a y e d b y a d d i n g D P N a n d a H~ s a t u r a t e d buffe~ to tile ~iLzyme. N o la K pha,se w a s f o u n d w h e n t h e e n z y m e w a s p r e i n ~ u b a t e d w i t h H s o r D P N H (Fig. 2). T h e l a g p h a s e c o u l d a l s o b e r e m o v e d b y i n c r e a s i n g t]',e c o n c e n t r a t i o n o f t h e e n z y m e {Fig. 2). O t h e r r e a g e n t s s u c h a s G S H , c y ~ t e i , e , a ~ : o r b a t e a n d f e r r o u s a m m o n i u m s u l f a t e w e r e i n e f f e c t i v e in r e m o v i n g t h e lag p h a s e . I t w a s p o s s i b l e t o increa~e t h e lag p h a s e 3 - f o l d b y p r e i n c u b a t i n g t h e e n z y m e w i t h o.x M NaC1, T h i s effect w a s n o t revers~,d b y t h e a d d i t i o n o f H a o r D P N I I . P l o t s w e r e m a d e a c c o r d i n g t o t h e m e t h o d o f LllqI~w~.AVER-BI2RI~ o n t h e effect o f D P N c o n c e n t r a t i o n o n its r a t e o f r e d u c t i o n b y Ha a n d t h e r e s u l t s s h o w n in F i g . 3 w e r e o b t a i n e d a t d i f f e r e n t c o n c e n t r a t i o n s o f H t. S u c h k i n e t i c s a r e c o n s i s t e n t w i t h t h e m e c h a n i s m f o r a t w o s u b s t r a t e s y s t e m in w h i c h t h e e n z y m e x i s t s i n t w o d i f f e r e n t states (oxidized a n d reduced) a n d the s u b s t r a t e can give one of the p r o d u c t s w h e n reacting with a particular form of the enzymetL Eqns. z and z represent such a m e c h a n i s m . ~VINFI~LI)17 h a s p r o p o s e d E q n . z for t h e m e c h a n i s m o f h y d r o g e n a s e , E + l[l~
EI|a~-~ E H -
"t- !! ÷
EH~ + DPN+ W E l i " DPN r~t E + D P N H
(I)
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B i ~ . k i m . B ~ o p l y s . At:a, f,7 (z963) 5 8 9 - 5 9 8
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concentrattl~n. I)|'N[! ,aT~(I 1-Jj ~bt, l a g pho-~'-~ t)f t l e h y d r o g ~ - l ~ a s e r e a c t i o n . C - u r g e I, o o 5 u t ~ i t o f e n _ ' y m t , p r c i n c u l J a t e r l - . v i . h c . i t h t , r [ ) P ~ ' q H o r H = a~; d e s c r i b e d i n T a b l e L. C u r e r : ~, tht~ s a m e ~-'~ C u r v e z except no DI'N}I (~r II z a d d e d re) p r t . i n c ~ , h , . l i o n mixt,re Fc, r C u r v e ~, u ÷ - ' . $ , n i t t)f et~z~,-r~le a n d C u r v e -I~ (~ z 5 ~lni~ z f e n z y m e w~re ,.~d.
taking a.~ his mc~(tel catah-tic hydrogt.nati,,n and the rt~ults Hf K ttA.~NA A.~D RITTENi ~ E R G It. The rate equation fiw thi~ m(,ch:ttaism i~ Eqn. 3 |'m,,,du
"-
t
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h',~t|l~
(3)
-..ICt~.,;/D|)N
whece v is initial velocit.v, l-",,~ m a x i m u m vel,~citv, Kuz, Kr)p.,; cc, nstartts and H~. D P N molar cortcen~ration o f [I~ and D P N . W h e n the LINEWEAVF.R-I~URK pl0t is m a d e with e q u i m , l a r concentrations o f sub.~tra'.~.~ which are varied simultaneously. f
:
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I
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4
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3 I/DPNX
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[~'iK. 3. l : f f ¢ c t n f |)lJ.%l a n d | 1 1 c o n c e a t : a t i t m on the ra[e of hydrogen dehydrosenase re;xctiot]. For C u , v e ]. t . 6 m l o f H t s a t u [ a t e d b u f f e r ; C u r v e z, z . z n d o f I - l ~ s a t u r a t e d I ) u t f e r ; C u r v e 3, 2 . 8 m l of H s -'~tturated buffer ~vcre used. Reaction .,,tartcd by ~.dding 0.03 unit of enzyme t o u..~ay mixture.
Eqn. 3 r e d u c ~ to a linear relation between the reciprocals o f initial velocity and substrate concentratio2~ (F~g. "1! Other typ~ : ,~f meehani.~m for t w o substrates w o u l d give a non-linear plot i°. 1J~o~Jli~ Biopllys .4eta. (~7 0 9 6 3 ) 5 8 9 - 5 9 8
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4. Rff,'c', of eq~imota( c t ~ n c c - n t r a t . i o n . . , o f ] ) I ~ N isn¢] H l i ) n t h e r a t e ll~ saturate,! buffer containe0 7.z • zo-* M I)['N
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2 ml of solution c o n t a i n i n g 5 u n i t s of hydtx>gen dehydrogenaae. 6/~nat~l<~ D e N a n d 2 o / , m o k s "u'ris-HCI bufl'~.r (pH 7.H) wt,re placer| ()n top of the gl&~.q .~ca[ of the via] which c o n t a i n e d a p p r o x . o.<~ l~,nolv t r i t i u m Ka~ (50 m('} in a.o ml. Tht: r~..-~t of the c o n t a i n e r was tiushed with helium a n d t h c : ~ ~ u a l c d with a gta.~.~ stopper. R ~ - ~ c t i t ) n staJrte,i by b r e a k i n g v i a l s e a l a n d i n c u b a t e d for t o rain. C(*tst*.nt~ w~.rt: r*,mt*w-*~, plac,~cl in a tuht~ c o n t a i n i n g 4 ° / , m o l e s D e N t | a n d t h e n heated to go e for rain. rt.~Lt~tion m i x ~ r ~ ¢oolt.d a n d centrifogeernatant was n e u t r a l i z e d with [ l~l KOI{. After the KCIO, had p r e c i p i t a t e d , t h e sul~crnttt;znt w;ts ttlplit~l t~ a 7 x 0.9 curt t:oh~mn of Dowex-t f o r m a t e |t a n d the c o l u m n wa~ t h e n wa,qht,tl with 50 ml of ~-att,r, D e N a.~tl th,, acitl ~'ere ehltc~l with a linear g r a d i e n t b~vtween 5o m | xt.';ttt~r *Xtltl 5<) t l ' t | 3 .~l f ~ ) r r t ~ j t " a t ' i ~ . l ; 2 - 1 1 1 ] f r ; t ( : t i o n s were eollectett. T h e g l u t a m i e d t ~ t y t l r t ~ g e n a s e re-'tzti(|f~ t~t'tixtur(: contaltxe
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iron for m a x i m u m a c t i v i t y o f t h e enzvme~, f r o m Clostridium tn~tylicu,n e a n d D. destdfuricans a. F e r r o u s iron is al.~o i m p l i c a t e d in s y s t e m s r e d u c i n g D P N 5.zn.m. W o r k cJn t%e~domonas saccharoflhila s u g g e s t s t h a t iron is linked to t h e p r o t e i n b y a. thiol g r o u p ~s. A fuller do.~rription of t h e b o n d b,.-i~lg split will r e q u i r e dat,n f r o m q u a n t i t a t i v e studie.~. O n r e d u c t i o n o f h y d r o g e n dehydrogetla.-~e ~f H. r~¢hlandii ~,,-ith II~ (~r D P N H , t h e b o n d is split a n d prt,duce~ a P C M D ~ ' n s i t i v e third ~rnup(s}. "[he lack o f inhibiti~m b y a r s e n i t e .~uggcsts t h a t a vicinial dithiol i.~ n(Jt l)r(,duced on r e d t l c t i o n <~f t h e e n z y m e . T h e r e d u c e d b o n d is a s s n m e d to r e a c t with. D P N to give D P N H , wllich w o u l d b e in a c c o r d w i t h c y a n i d e , sulfide a n d sulfxtt. :wting as ~ ' o m p e t i t i v e i n h i b i t o r s o f I ) I ' N (Fig. z). K i n e t i c a n a l y s i s al-¢.o s u p p ~ r t s this t y p e of m e c h a n i s m i n w ) l v i n g t h e r e d u c t i o n - - o x i d a t i o n o f r. s u l f u r b r i d g e a n d h a s b e e n f<~rmulated as F.qns. x a n d 2. T h i s m e c h a n i s m c o u l d e:q)lain t h e inhibit<~r,," :t(-tion o f o x y g e n on h y d r o g e n a s e b o f a n a e r o b i c b a c t e r i a w h i c h ha~ p r e v i o u s l y be~:n i n t e r p r e t e d as o x y g e n a t i o n a n d o x i d a t i o n of t h e enzywne2L O x y g e n a t i o n inhibiti,~n w a s foui,d t',J he r e v e r s e d on r e m o v a l o f ( , x y g e n t7 a n d Off:, i n h i b i t i o n c a n ntJw t),. a c c o u n t e d for as t h e o x i d a t i o : l o f t h i o i g r o u p l s) w h i c h axe g e n e r a t e d on reducti()n ()f t h e e n z y m e b y H.~. T h e r e is a n anal¢~gou~ e n z y m e , t h e fla~-opr~,t,-~t d i h y d r o l i p o y l d e h y d r o g e n a t e , w h i c h sho~vs t h e s ~ m e k i n e t i c s ~ as Fig_ 3 fi~r h y d r o g e n d e h y d r o g e n a s e a n d h a s a di~ulfidexlithiol g r o u p as an i n t e r m e d i a t e ~.n~. T h i s f l a v o p r o t e i n aL,~o c a t a l y z e s a n e x c h a n g e r e a c t i o n b e t w e e n I ) P N H anti d , : u t e r i m n o x i d e ~. S e v e r a l o f t h e flavop r o t e i n d e h y d m g e n a - , ~ s s h o w v a r y i n g e x c h a n g e r a t e s 2~.~2.m a n d in one c a s e C~ere ~s n o e x c h a n g e za. F l a v o p r o t e i n s a r e n o t u n i q u e in pr~)moting a n exchanT~'c• b e t w e e n a r e d u c e d s u b , i r a t e a n d w a t e r . T h e l|ytlrogena~e ~f D. des~tlfuricaits c a r r i e s o u t a n e x c h a r t g e b e t w e e n t h e e n z y m e h y d r i d e a n d d e u t e r i u m oxide, c~)ncurrently w i t h t h e r e v e r s a l n f a r e a c t i o n like E q n . ~. T h i s r e s u l t s in t h e a p p e a r a n c e o f t w o species o f h y d r o g e n i,~. D D a n d H D a n d t h e r e l a t i v e ratt. {)f fi~rmation o f D D t(~ H D is x.x { ~ e ref. 2}. T h e expe.'-iments w i t h t r i t i u m isot{)pe a n d h y d r o g e n d e h y d r o g e n a s e d o not distinguish between reacti~n x and the exchange between the enzyme and water. As t r i t i u m is i n c o r p o r a t e d r a p i d l y i n t o D P N H f r o m H T O , it c a n be c(~ncludeti f r o m t h e appeara~ace (ff l a b e l l i n g o n l y in the/q-l~)sition o f I ) P N H, t h a t t h e s t e r e o s p e c i f i c i t y o f t h e e x c h a n g e a n d dehydrogena.~., r e a c t i o n ~ arc t h e s a m e . T h e r e m o v a l (~f t h e lag p h 0 s e b y p r e i n c u b a t i n g t h e enzxa'~e w i t h 14: or D P N H c o u l d lm e x p l a i n e d b y t h e e x i s t e n c e o f t w o e ~ z y m i c t'orms, o n e being i n a c t i v e w h i c h is in e q u i l i b r i u m w i t h t h e a c t i v e f o r m . O n reductit,~a o f t h e a c t i v e f o r m , t h e i n a c t i v e f o r m is s l o w l y c o n v e r t e d i n t o t h e a c t i v e f o r m . Thi~ lag pha_ee c o n t r a s t s w i t h tit~,t d e s c r i b e d b y ~VIT'I'E?:BERC, AND R~.r,,~SKE ~s for e x t r a c t s o f H. eulropha w h i c h red u c e d I ) l ' Y w i t h H t a m i h a d a lag p h a s e t h a t wa:" n o t a f f e c t e d b y D P N H or Hz. T h e effect o f ~ulfide alsn d i s t i n g u i s h e s t h e I ) P N r e d , I c i n g .~.,stem:; in hi. eutr~pha a n d H. ruhla~tdii w h e r e in lht' f,~rmer sulfide a t i r r ~ l a t e ~ a c t i v i t y 3e a n d in t h e l a t t e r is a s t r o n g i n h i l , i t o r ( T a b l e II. ACKNO~VI,I:,I)t',E~II".N 1.'4
T h i s w o r k wa~ s u p p o r t e d b~ g r a n t s f r o m t h e N a t i o n a l I n s t i t u t e s ~,f H e a l t h a n d t h e N a t i o n a l .%cience Foundation;. T h e a u t h o r wishe~ to t h a n k Mr. A. ]~OSENBERG o f t h e C h e m i s t r y . D e p a r t m e n t . Y a l e U n i v e r s i t y f , r t h e a~..~ay o f t r i t i u m g a s i~t t h e e x c h a n g e experiment with tritiated water. f~!c, ch:m. 13~ophys÷ .4ct~. 67 (Iq(,3} 389 5q8
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REFERENCES If. H . | ) . HOBI~RM&N ANI) Do I~ITTKNI~I£RO, J . B i o l . C h e m . . *47 (*g43} 2 [ ! . * A. I. KR^StCA. E. R t X L t s xNr~ D. RITeRtCRr.~C. J . B i o L C k e m . . 235 ( ] 9 6 0 ) 2 7 , 7 ,
• D. R t ~ r ~ N B E R G . P r o c e e d i n g s o f t h . I n t e r n a t i o n a l S y m p o s i u m a)t~ K y o t o , z 9 5 7 , lCi&ruzctx a n d C o . . T o k y o , z 9 5 8 . p. 2 5 0 .
on E n z y m e
Cl~emistry.
Tokyo
* E . H l ~ c l s x ~ v D, R t T - r t : , B ~ R G . ] . B:'#L C h u m . . ~36 ( , 9 6 t ) z 5 2 6 . K. M^cc.~y, j'. B w l . Chem., 236 (Iq6x) 574. • El. D. PECK. JR. ANO H . G~:sa', J , Ba, ierivl., 73 ( 1 9 5 7 ) 569. 7 A . L. S~tuG, P. %V. V¢]L~ON. D. E . G a E B N ^ N D H . R. M ^ H C Z R . J . .din. Chem. Soc., 76 ('954) 3355, t j, C. ~,%DANA AND A. V . MORLEY, Bgocait~qt. ~JiopJty$..~Ctfl;, 5 0 ( I 9 6 1 ) 153. t A. | . KK~.~::';~. AND D . R1YTENBERG, J . ~ m . C h e m . So&., 70 ( r 9 5 4 ) 3ol.5. ie 13. H - HONE, S. BB-qNSTEIN AND ~t~'. VICtHNIJt.C, BiocAim. B t o p / e y s . A c I a , 67 (1963) 5 8 1 . ,, R C ~ , / L A , t , J. R . M C P . R X , i . c B. A N F , , , ~ E N , J R . . M. L . A N S O N . K. BAILEY A N D J. T . EIJe~ALL. Mdv~tn. P r o t e i n C h e m . . V o l . 14. A c a d e m i c P r e s s . I n c . . N e w Y o r k , *959. p . 1 3 6 . i, M. D i x o n ^Ni* E+ C. XVEBB, E~caymes. L o n g m a n s , G r e e n s n d C o . . Ltd÷. L o n d o n , ] 9 5 9 , P. 374. t• H- T~'vt^%-~2~it, ~r~t~ C. S. VESTLIN6, B i o ~ h i m . B t o p h y , . A ¢ t a , 2 0 ( I 9 5 6 ) 5H6. ;a j. C. SADAN& AND V. JAGX.~NATHAN. B i o c h i m . B i a p k y s . . , I o t a , *9 {t~56) 44 o. *~ S. BL,~Cx ,,St) B. H U n S O N . Bio~hem. BiopAys. Res. C o m m u . . . 5 (*O6t) t 3 5 . at R . A. ALZtI-.RX'V, ill F . F . NOSD, A d v a n . E u ~ y m o L , V o l . ] ? , i n t e r s c i e n c c , Ne~. Y o r k , g 9 5 6 , p . t. it i . E . %VINFIELD, Rg~t. P u r e ~ p p l . C~14~m., 5 ( I 9 5 5 ) 217" x.. Ho Ft. I.F.VY AND ]~. VENNIK~LAND, J . Biol. C h m . , 2 2 8 ( I 9 5 7 ) 85. xt j+ I.. ~ R A V ~ S , O. V I ~ N N ~ L A N D . M. I r. U T t E ~ AND ]{. J . ]*ENN|NGTON, J . B i o l . ~Ml~g., 2 a 3 (195 I~) 551. t* T . ]~rAKAbtP*fO AND ~ . X:ENNIZ.SI.AND, J . Biol. C h ~ . . 2 3 5 (19fl4~) 2 o 2 . | t B. K. STF.RN AND H. VENNESLAI~O, J . Biol. C a i n . . 2 3 5 (tO6o) - o 5. | I B. K . STt:RN AND B. V E N N E S L A N I ) , J . BiOl. CAem., 235 (x96o) ~o9. "* ",..A. H:":u~::,':. P,. H . ~t+~.i~xt~ ,~ND P. Me. %%',L50.'% J. B ~ r ~ l . . 63 (~953) 5z~+ : t | . ~'RIDOVICH AND 1). H A N D L E R . J . Biol. Chem., 2 3 I ( | 9 5 8 ) ~l'O9. s* ! ) H. 14oNE. u n p u b l i s h e d o b s e r v & t i o n s , tl~ ]~. I~I~PAf~KK AND C. SE:%%'&RD, B a c t a r i o L J~oc., ( 1 9 6 1 ) 168, :" 1{. V. FIS){ER, A. ] . KRASN'A AND D . R I ~ E N B E R G , J . B ~ . C ~ m . , 209 [ 1 9 5 4 ) 5(~t) =s H . B c 3 ? w . R. B . HURLBER'r ~ q D V. R . POTTER, J . 85ol. C h e m . , I 9 6 (195~) 717 • t t "%' MASSEY, Q H GIBSON AND C. VElgGER, Bic~.hem. f . , 77 (IO6O} 3 4 ' ".u V. M ^ s s E V XND G. V~'EGKI~, P . i o c h i m . B i o p h y s . A e t a . 48 ( x 9 6 * ) 33. )I ~ . I,. SE.%RL~. J . M. P E T E R S AND D . R . SANADI, J , Bi01. C$14$)$1., 2 3 6 ( x 9 6 t ) 23* 7. '* M. M. %%'zr~tt, N. O. K*,PI,^N, A . SAN P I I ~ R O X.','D F . E , S1rOLZI~NBACll. J . Miol. C h e r t . . ~ 7 (t957) :7a~ G,. I(. DRYSDALE. J . BIG:. C h e m . . 234 ( t 0 5 9 ) 2 3 9 ~ . a.t (;. }.~. ])RYGDAL~-'. M. J÷ ~ P ' E G E L AND P. ,qTR*T~m^X"rER, J . F3ioL C h e m . , 2 3 6 ( * 9 6 t ) 232:3. at C. I, V~'ITTEN)I1M.RC~A~t'J R ~I~.VAS~I~, B ~ c h i y n . B w p h y s . . 4 e t a , 47 ( * 9 6 z ) 5 4 2 . R÷ RKI'ASKb., J÷ / J i l l . C h e m . , 237 [1~32J I 3 3 I . " S. C. K I N ~ K Y , E. R, S'IIADTMAN .~ND r{.
B i o c h t m . B=ophys+ .4,ta. 67 ( 1 9 6 3 ) 5 8 9 - 5 9 8
~9
neA z2x59
INHIBITOR, ISOTOIqC AND KINETIC STUDIES ¢)N H Y D R O G E N D E H Y D R C b G E N A S E D. ][. i$ONK • Deparlm¢):t ~ f ,a,Z icrc'~.i,plo;:y, $'¢*l¢ L; )r it', r.set),, Neu, H a v e n , Co))n. ( U..g..-I .)
( l t e c e i v e d A u g u S t z 7 t h , nqt,.,.)
SUMMA L,,L Y T h e oxidized form of h y d r o g e n dehydrogena_se (,f H vdrogt'~tzomo~:as rJrJdamtii was in* hibited b y BAL. cyanide, dithianite, sulfide. ~uifite a n d thioglycolate. Reduction of the d e h y d r o g e n a s e with DPN H or I'I~ resulted in a dindnished effect of the inhibitors, w i t h the exception o f p-chiorome=curibenzoate which inhibited only the reduced e n z y m e . I n f o r m a t i o n from kinetic analysis and inhibit(~r studies indicates t h a t the e n z y m e is reduced with H , to give a proton and t h a t this i~ followed by the oxidation o f t h e reduced e n z y m e w i t h D P N to gi;'e DPN H. The enzyme catalyzes an exchange between H~ a n d H T O a n d also between HTO a n d D P N H , Analysis of DPN enzymically reduced with T a shows t h a t 1" is in the//-position (,f DPN H. The e n z y m e actic~n h a s a lag phase which (~an be eliminated b y preincubation with I-I~ or D P N H . llgTICt)[~UCTION
T h e m e c h a n i s m o f action of h y d r o g e n a c t i v a t i n g en/.yme~ has been investigated from the viewpoint of exchange reactions u,2 the metal comlx)nenO,' and a possible flavin comportentr-L Th," hydrogenase o f Desulfovibrio destdfi~rlcav.s has been e x t e n s i v e l y investigated :.4.~' a n d RaKI.Is ,x~i) R r ~ E x ~ r t 6 ~ have o b t a i n e d a highly purified preparation using the exchange reaction betwL~en deut,:rium oxide am] h y d r o g e n as the a s s a y system. The hydrogenase of D. desulfi,Hcans can aIso re~luce benzyl viologen, m e t h y l e n e blue a n d liberate h y d r v g e n fn)m reduced m e t h y l vioIogen*, a. Spectrographic analysis o f this protein showed iro,~ to be the major metal component. The exchange reaction indicated t h a t the hydrogen;~se reaction can occur as a heter(flytic proce.~ resulting in the formation of an enzyme h y d r i d e and a proton ~. H y d r o g e n d e h v d r o g e n a ~ of Hydrogenomonas ruhlandii provides a more amenable system for the s t u d y o f the activation o f hydrogen and in this report a t t e n t i o n has been focus.ged on inhibitors which cat) react with disulfi~le groups, the u~e of i-~otop,~.~ to evaluate the exchange reaction.~ and a kinectic analysis of the h y d r o g e n d e h y d r o g e n a ~ reaction. A b b r e v i a t i o n : I-~CMLL p-chluromecuril~.'nzo~Lte• I~reoent a d d r e s s . D e p a r t m e n t o f B o t a n y . Q m ' o n M a r y College. U n i v e r a i t y o f L o n d o n , L o n d o n E . z ( G r e a t lJritain)÷ t4toch;m, i d i o p h y s . . 4 c l a . 67 (1063) 5rig-.508
5fjO
D.H.
BONE
MATERIALS AND METHODS
T h e f.)llowing m a t e r i a l s w e r e c o m m e r c i a l p r e r ~ a r a t i o n s : p i g - h e a r t m a l i c d , : h y d r o g e n a s e {C F. B o e h r m g e r a n d .qohne, M a n n h e i m , G e r m a n y ) , l i v e r - g l u t a m i c d e h y d r o g e n a ~ (Sigma C h e m i c a l C o m p a n y ' ) , D P N a n a l o g s ( P a b s t L a b o r a t o r i e s ) . I l y d r o g e u d e h y d r o g e n a s e u s e d w a s e n z y m e B a n d a s s a y e d as d e s c r i b e d in t h e a c c o m p a n y i n g patx:ra°. K i n e t i c m e a s u r e m e n t s w e r e m a d e using o . x M T r i s - H ( ' l buffer ( p H 7-8) m t u r a t e d w i t h H~ a t 25 °. T h e s o l u b i l i t y o f H t in Trig b u f f e r is a.~sumed to be t h e s a m e as in w a t e r . T h e t e c h n i q u e for t h e m e a . ~ u r e m e n t ~f r e d u c e d a n a l o g s of I ) P N is t a k e n f r o m t h e P a b s t L a b o r a t o r i e s C i r c u l a r O R - x S . S a m p l e s o f t r i t i a t e d m a t e r i a l w e r e p i p e t t e d o n t o p l a n c h e t t ~ a n d t h e n d r i e d d o w n E a c h .~ample w a s t a k r n u p in 0.2. m l o f w a t e r a n d e v a p o r a t e d t o d .r3,ne~g 5 times. R a d i o a c t i v i t y w a s t h e n m e a ~ x r e d u s i n g a windowle~q gag flow c o u n t e r . RESULTS
I nhibdor.s T a b l e I s h o w s t h e effect o f i n h t b i t o r s on h y d r o g e n d c h y d r o g e n a s e . T h e i n h i b i t o r s listed are r e a g o n t s w h i c h split dis ."tale b o n d s n a n d a]so can, in t h e c a s e o f c y a n i d e a n d s u t h d e r e a c t w i t h a m e . t a P a. T h e r e a c t i o n o f c y a n i d e a n d .~,ulfide w i t h t h e o x i d i z e d e n z y m e i~ a slow reactior, a n d t h e m a x i m u m i n h i b i t i o n is a t t a i n e d o n l y a f t e r a 5-rain i n c u b a t i o n p e r i o d w h e r e a s sulfite r e a c t s ~ n s t a n t a n e o u s l y w i t h t h e e n z . v m e a n d TABI.E F2FF/~I2T O F I N I I I B I T O R S
1
ON MYDRO4",RN DE~VDROGENASF.
ACTIVITY
l'rc~ncubAt~d solution.~ contninexl Ip'drogen dehydtoguna~ t~ufficient enzyme to give an t~bsorba u c y c h g n g e a t 34 ° m p o f o . z t r n i n in t h e f i n a l v o l u m e ) , adtt| 2 o / ~ r n o l e s T r i g | [ C l b u f f e r { p H 7.8}. For DPNII preincubation, the m,hnion contained enzyme. 2o/~rnoles "l'ns-succinate (pH 6.9) a n d o o b / c r n o l e L ) P N H . I n h y d r ~ g t . n p r e i n c u b a t i o n e x p e r i m e n t s , h y d r o g e n ,a a s Fm,sstx| t h r o u g h the solution. Prclncubatlon w a ~ .5 xnin a t z 5 ~. v o l u m c o f s o l u t i o n t,.2 m l a n t i c o l ~ c e n t r a t i o n o f i n h i b i t o r s a s in
I~*h~ic~n
t. %~
H I
(>
DPNI! ]c -t M xo - a M ~o "'l M ,o-'M to aM 5 ' to * 5.lc-4M 5 to-4 5" to" * .5"' t o - 4
K C N ( p H 7.8) K ( ' K (1,H 7.8) + KCIK { p H 6 . 9 ) K C N { p H 0.9~ + K C N (pH 7.8) -M N a t S (I', I 7.8) N a t S ( p { 7.8j M N a t S ( p H 6.9) M N a t S ( p H b-9} M N a t S ( p H 7.8}
o 4°
lit
7
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g7 4° 44 loo 4(i too 73 too
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5 " ~o-~ M N g t . q ( p H 7.8") 1o +a M N a t S O a ( p H 7.8) IO -a M N a t . S O , ( p H 7.8) + H s 5 " ] ° ' s M N,'htSsO a 5" t o - i M N a z S a O , 4- | ' I s 3"lo*M BAL 3 1o-* M B A L ~. I t e 5 " xo-~ M T h i o g l y c o d a t e 5 • t. i M T r . i o g l y c o l l a t e 4- H s 7"z, -M t'CMU 7"1o-¢ M I~CMB ¢ l| t
iflJl|bl$|0~ f%)
52 45 o 68 37 8a .56 *. 3 ~tj o 53
" t o -a ,'g{ C,.q~C. pfcs~2nt in rcac'~ion n11xtlJru. ..
Bio~him.
Fl:optD's..-Iota.
67
(1963) 5~9-598
FI~'I)ROGEN DEHY!)R|Jt;EN:kSE
59~
t h e r e a c t i o n is ~ompLet,.. in 30 ~ec. A t t e m p t s t(~ r e v e r s e ttJa_~ i n h i b i t i o n s o f N e z 5 ~_ad K C N b y (a) d i l u t i o n t h i r t y - f o l d witl, "l'ris buffer (b) r e m , , v a l o f i n h i b i t o r b y 5-'pha~lex G - z 5 gel raider conditirDns wtfi~-h efftn'Zivelv r e m o v e sulfide ( T a b l e 1) (cl dialyai.~ a g a i n s t t o o v o l u m e ~ o f o.05 M pota.~sium phosphatt~ ( p i t 7.,~). o.,~oz M E D T A o.o~Jqr/M MnCI. for z 4 h (d) treatmer,-: w i t h t~.ooz 31 ZnSO~ (e) additi,~,n o f 0.03 3I i x ) l a s s | u r n s a l t o f E I ) T A , w e r e not ~-ffcctix-~. T h e o n l y c o n d i t i o n w h i c h varti~:liy rev e r s e d t h e i n h i b i t i ( m o f sulfide wa.~ t h e d i l u t i o n t,t' t h e p t e i n c u b a 0 u t a m i x t u r ( . ,v'~eh a s o l u t i o n ()f (~%5(_; ( T a b l e I}. A i ) P N - s u l f i d e e n z y m e c o m p l e x h a s I-,~.~.,~ rep..~rt,~d a s t h e m e c h a n i s m ,~t"i n h i b i t i o n o f !:.,~th: ( l e h y d r o g e n a , e ~ a ; b u t in t h e case ~f h y d r o g e n d e h y d r t ) g e n a s e D P N h a d r.~ effect on tlk- inhibiti,,n ~f t h e , , ~ z y m e b y 2 - z o ~ M NaaS. F u r t h e r , D P N w a s f ~ u n d to h a v e nc) effect ~,~ ,tnv ()f t h e inhibitor~ e x c e p t in t h o s e k n o w n cu_ses when" ! ) P N r e a c t s d i r e c t l y w i t h the irthil)it~)t÷ T h e r e a g c n t ~ w e r a all n-,c,l~- ~fft-~tiv,: wht.l~ tht- i n h i b i t ~ r wa~ preie~c,~bated ~'~th t h e e n z y m e a n d the teacti~)n ~ t a r t c d b y a d d i n g I 2 : a n 0 I ) P N t h a n w h e n t h e e n z y m e w a s first r e d u c e d b y I] z or D P N H a n d t h e inhibilt~r t h e n a d d e d T h e e x t e n t tn ,vhich t h e p r e t r e a t m a n t a f f e c t e d t h e inhibitio.~ w..s ( l e p e n d e ~ t ~,n t h e i n h i b i t o r ( T a b l e I). V a r i a b l e r~-~;ult.~ fo- cynic|de i n h i b i t i o n h a v e i~.t.n r e p o r t e d for hydr~,~enase.~ f r o m a n a e r o b i c ba~teria~-'~.e.a. ~. T h e at~thor h~t~, t2~und r ~ a t ~i i~ e.~ct~tial to ~ t a n d a r d ize c o n d i t i o ; : s ft~r all i n h i b i t o r studie., a n d t h a t ~l_~t.~-ial c a r e be g i v e n to the. o x i d i z e d r e d u c e d s t a t e o f t h e e n z y ' m e , if e a r | a i s l e rcsult.~ are to he e l i m i n a t e d . I t w a s p o s s i b l e t o c a r r y ~)ut a r e d u c t i o n o f hyclr~gen d e h y d r o g e n a t e w i t h a s m a l l a l n o u n t o f H~ (0.3/~mole) to g i v e a n e n z y m e w h i c h is i n h i b i t e d 4-F'~'~, b y 5 " x o ~ M Na~S. W h e n t h e e n z y m e w a s tht:n o x i d i z e d w i t h 3 ~um~)le~ o f I ) P N a n d t h e nu~:let)tides f e i n t ) r e d w i t h S e p h a d e x G - 2 5 gel, a n , : n z y m e w a s o b t a i n t . d w h i c h is c o m p l e t e l y inh i b i t e d b y sulfide. P C M B i n h i b i t i g n v~tries in the r,:verse m a n m : r to t h a t o f ti~e o t h e r i n h i b i t o r s . t h e r e d u c e d enzxa~ne b e i n g m o r e s e n s i t i v e to P('.'~ B ( T a b l e I). A .~imilar effect w a s n o t i c e d w i t h glutathi~H~e reducta.,~, which bec'~me~ ~en~itivc t() ~ulth3"dryl r t . a g e n t s w h e n r e d u c e d w i t h T P N II o r D P N H ~. A n , ) t h e r inhibit,~r .~tu(t~t.d was xo ~ 5I a r s e n i t e w h i c h i n h i b i t e d t h e e n z y m e ~e"i~ w h e t h e r t h e et~z)'nl~, xwle, ~xidizcd or r e d u c e 0 . T h e effects o f v a r y i n g t h e c o n c e n t r a t i o n t)f N a t l . u r e a a n d T r i s - H C l bufft~r (pI-t 7.Y) w e r e t r i e d in a r s e n i t e i n h i b i t i o n e x p e r i m e n t s using e n z v m t . s A a n d pl~0 a n d enzvn~e B p r e p a r e d in t h e a b . ~ n c e o f m a n g a n e s e . I n n~) case w a s t h e i n h i b i t o r y effect ()f a r s e n i t e |not'eased. O x y g e n w a s f o u n d ~ o t to inhibit t h e e n z y m e a n d nn precaution_~ w e r e f o u n d n e c e . ~ r y tt~ e n s u r e t h e a b e - , ~ o f o x y g e n d u r i n g t h e purilicatic)n pr(~cedure t°. A s sulfide m a y fi)rrn a s t a b l e c o m p o u n d w i t h t h e e n z y m e , the e n z y m e (~oo ~g) w a s i n c u b a t e d w i t h NaaaaS ( o . r / a m o h : , 2 - x o ~ eount.~;mit~) for zo rain a t r o o m t e m p e r a t u r e . T h e sulfide a n d e n z y m e w e r e s e p a r a t e d b~" t h e a d d i t i o n o f sufficient .~aturated (NH~).,qO, st)lution t~ b r i n g the s a t u r a t i ~ m to 6o0~. T h e p r e c i p i t a t e d p r o t e i n w a s w a s h e d t w i c e w i t h 6 o ° ~ saturat,~d (N[I.,)~S() a a n d its r a d i o a c t i v i t y t h e n m e a s u r e d . N o t-ad:'oactivity w a s a s s o c i a t e d w i t h t h e p r o t e i n .
tginelics of inhibited enzyme Tile e f f e c t s o f t h e inhibitt~rs c y a n i d e , sulfide :rod ~ulfite w e r e i n v e s t i g a t e d b y m e a s u r i n g t h e r:Lte-n o f r e a c t i o n in the p r e ~ m c e ()f a c o n s t a n t a m o u n t o f e n z y m e , i n h i b i t o r a n d e x c e , ~ .~uh~trate, whilst v a r y i n g t h e c o n c e n t r a t i o n o f t h e o t h e r s u b Bi.ch~m l~ophys..4ct~. /J7 ('(c)63) 589-598
D . H . nONE
592
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!
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k~ D
~oc
o
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ec
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Fig. ]. Effect of cyanide, sulfide and sulfite on hydrogen dehydrogenase. 0.0 4 unit of oxidized enzyme preincubat~_~l ~ in Tahle I with ]o -i M KCN (Curve a). to -a M NaaSO. |Curve 3) and z-5 ÷xo -¢ M Na,S (Curve t)- Reaction started by adding varying amounts of D P N and H a saturated buffer.
s t r a t e . T h e L i n e w e a v e r - B u r k p l o t w a s u s e d t o a n a l y z e t h e r e s u l t s (Fig. t). All t h r e e i n h i b i t o r s were c o m p e t i t i v e w i t h D P N a n d p a r t i a l l y n o n - c o m p e t i t i v e w i t h Ha.
Kinetics of red,ction of D P N ~_ hydrogen A lag p h a s e w,~Ls . h ~ e r v e d w h e n t h e e n z y n l e w a s a s s a y e d b y a d d i n g D P N a n d a H~ s a t u r a t e d buffe~ to tile ~iLzyme. N o la K pha,se w a s f o u n d w h e n t h e e n z y m e w a s p r e i n ~ u b a t e d w i t h H s o r D P N H (Fig. 2). T h e l a g p h a s e c o u l d a l s o b e r e m o v e d b y i n c r e a s i n g t]',e c o n c e n t r a t i o n o f t h e e n z y m e {Fig. 2). O t h e r r e a g e n t s s u c h a s G S H , c y ~ t e i , e , a ~ : o r b a t e a n d f e r r o u s a m m o n i u m s u l f a t e w e r e i n e f f e c t i v e in r e m o v i n g t h e lag p h a s e . I t w a s p o s s i b l e t o increa~e t h e lag p h a s e 3 - f o l d b y p r e i n c u b a t i n g t h e e n z y m e w i t h o.x M NaC1, T h i s effect w a s n o t revers~,d b y t h e a d d i t i o n o f H a o r D P N I I . P l o t s w e r e m a d e a c c o r d i n g t o t h e m e t h o d o f LllqI~w~.AVER-BI2RI~ o n t h e effect o f D P N c o n c e n t r a t i o n o n its r a t e o f r e d u c t i o n b y Ha a n d t h e r e s u l t s s h o w n in F i g . 3 w e r e o b t a i n e d a t d i f f e r e n t c o n c e n t r a t i o n s o f H t. S u c h k i n e t i c s a r e c o n s i s t e n t w i t h t h e m e c h a n i s m f o r a t w o s u b s t r a t e s y s t e m in w h i c h t h e e n z y m e x i s t s i n t w o d i f f e r e n t states (oxidized a n d reduced) a n d the s u b s t r a t e can give one of the p r o d u c t s w h e n reacting with a particular form of the enzymetL Eqns. z and z represent such a m e c h a n i s m . ~VINFI~LI)17 h a s p r o p o s e d E q n . z for t h e m e c h a n i s m o f h y d r o g e n a s e , E + l[l~
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2 ml of solution c o n t a i n i n g 5 u n i t s of hydtx>gen dehydrogenaae. 6/~nat~l<~ D e N a n d 2 o / , m o k s "u'ris-HCI bufl'~.r (pH 7.H) wt,re placer| ()n top of the gl&~.q .~ca[ of the via] which c o n t a i n e d a p p r o x . o.<~ l~,nolv t r i t i u m Ka~ (50 m('} in a.o ml. Tht: r~..-~t of the c o n t a i n e r was tiushed with helium a n d t h c : ~ ~ u a l c d with a gta.~.~ stopper. R ~ - ~ c t i t ) n staJrte,i by b r e a k i n g v i a l s e a l a n d i n c u b a t e d for t o rain. C(*tst*.nt~ w~.rt: r*,mt*w-*~, plac,~cl in a tuht~ c o n t a i n i n g 4 ° / , m o l e s D e N t | a n d t h e n heated to go e for rain. rt.~Lt~tion m i x ~ r ~ ¢oolt.d a n d centrifoge
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a(~ u-.~teret~l>U-¢:ili<: e n z y m e TM. l t ~ t d i o : i c t i v i t y w;t.~ f o t m d i n g l u t a m i c a c i d a n d i n D P N produc('d in the malic dehydrogenase reaction {Table IV) -vhich means that hydrogen d e h y d r ~ g e n a ~ s e ha.', fl-stt~reospecificit3;. T h i s m e | h i n d (>f u s i n g a - a n d f l - s t e r e o s p e c i f i c er~z's'me.~ t o d e t e r m i n e t h e .,,tereo.~pecificity o f a d d i t i o n o f h y d r o g e n t o T P N h a s b e e n a p p l i e d t o se~'ura] e ~ u z ) ' m ~ :a t-°. I)I~CUS.~ION ( ~ y a n i d c h a s htx.n t i l e <>nix- i i i h i t > i t o r ~)f h y d r o g e n a s e s w h i c h h a s b e e n s t u d i e d b y . ~ v , . r a l wa)rk~,rst.2.a.0.n*, za, C y a n i d e h a s b e e n s h o w n t o i n h i b i t t h e e x c h a n g e r e a c t i o n o f t h e h~'drogenat.~t: o f Pro|errs ",,ulgaris b u t o n l y w h e n t h e e n z y m e is o x i d i z e d I-*. .%.imilarly, tht: oxittiz~:d f~)rm t~f ..|¢olobacler t,i~lelapndii h y d r o g e n a s t : is i n h i b i t e d b ~ t~v,anid~ z~. ~.%"ith h i g h l y p u r i f i o d p r t ' p a r a t i o n s o f h y d r o g e , t a s e f r o J n / ) . d e s t d f u ~ ' i c a n s thq~r~ t'xist.~ ~t tti.-,crel)~mt:~- i n t h e e f f e c t s o f c y a n i d e S , t*. T h e s e d i f f e r e n c e s c o u l d b e i t t t r i b u t e d t o tht, d i f f e r e n t a.,;.~a.~,, c o l , d . i t i o n s . (a) E x c h a n g e ~ ' , : a c t i o n a t p H 6 . 7 : c y a n i d e (xo a M) i n h i b i t s the_ o x i d i z e d f o r n l 25' ot,.o (see ref. 2). (b) M e t h y l e n e b l u e a s av, h y d r o g e n a c c e p t o r ,,t p H 2~.4: t:y~tnide ( z o -a M) i n h i b i t s zoo~)~,, i t , d e p t ; , , d e n t o f t h e s t a t e or" t h e e n z y m e , i . . (*×ittizcd t)r r e d u c e d it. "l'ht, m ~ r e d e t a i l e d .',tudie.n w i t h c y a n i d e a n d o t h e r i n h i b i t o r . ~ o n h y c h - o g e n d e h y d r o g e n a s e c,Y H . r:~hlan~tii ('l'atfle I) i n d i c . a t e t h a t t h e i r e f f e c t is l i k e l y t o b e d u e t<~ t h e . ~ p l i t t i n g c>f e i t h e r :x d i s u l f i d e b o n d o r p e r h a p s a m e t a l s u l f u r b r i d g e s u c h a s is i x > s t u l a t e d f o r x a n t h i x e o x i d a s e t~. T h a t Jr'on is p a r t o f t h e m e c h a n i s m o f h y d r o g e n a c t i v a t i n g e n z y ' m e s h a s br, e n i n d i c a t e d b y t h e p r e . ~ e n c e o f i r o n i n a h i g h l y p u r i f i e d p r e p a r a t i o a (.f h y d r o g e n a s e ~)f D. desulfu~ica~t.s ¢ a n d b y a r e q u i r e m e n t f o r f e r r o u s
B~ach~tn Biopkys..4
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iron for m a x i m u m a c t i v i t y o f t h e enzvme~, f r o m Clostridium tn~tylicu,n e a n d D. destdfuricans a. F e r r o u s iron is al.~o i m p l i c a t e d in s y s t e m s r e d u c i n g D P N 5.zn.m. W o r k cJn t%e~domonas saccharoflhila s u g g e s t s t h a t iron is linked to t h e p r o t e i n b y a. thiol g r o u p ~s. A fuller do.~rription of t h e b o n d b,.-i~lg split will r e q u i r e dat,n f r o m q u a n t i t a t i v e studie.~. O n r e d u c t i o n o f h y d r o g e n dehydrogetla.-~e ~f H. r~¢hlandii ~,,-ith II~ (~r D P N H , t h e b o n d is split a n d prt,duce~ a P C M D ~ ' n s i t i v e third ~rnup(s}. "[he lack o f inhibiti~m b y a r s e n i t e .~uggcsts t h a t a vicinial dithiol i.~ n(Jt l)r(,duced on r e d t l c t i o n <~f t h e e n z y m e . T h e r e d u c e d b o n d is a s s n m e d to r e a c t with. D P N to give D P N H , wllich w o u l d b e in a c c o r d w i t h c y a n i d e , sulfide a n d sulfxtt. :wting as ~ ' o m p e t i t i v e i n h i b i t o r s o f I ) I ' N (Fig. z). K i n e t i c a n a l y s i s al-¢.o s u p p ~ r t s this t y p e of m e c h a n i s m i n w ) l v i n g t h e r e d u c t i o n - - o x i d a t i o n o f r. s u l f u r b r i d g e a n d h a s b e e n f<~rmulated as F.qns. x a n d 2. T h i s m e c h a n i s m c o u l d e:q)lain t h e inhibit<~r,," :t(-tion o f o x y g e n on h y d r o g e n a s e b o f a n a e r o b i c b a c t e r i a w h i c h ha~ p r e v i o u s l y be~:n i n t e r p r e t e d as o x y g e n a t i o n a n d o x i d a t i o n of t h e enzywne2L O x y g e n a t i o n inhibiti,~n w a s foui,d t',J he r e v e r s e d on r e m o v a l o f ( , x y g e n t7 a n d Off:, i n h i b i t i o n c a n ntJw t),. a c c o u n t e d for as t h e o x i d a t i o : l o f t h i o i g r o u p l s) w h i c h axe g e n e r a t e d on reducti()n ()f t h e e n z y m e b y H.~. T h e r e is a n anal¢~gou~ e n z y m e , t h e fla~-opr~,t,-~t d i h y d r o l i p o y l d e h y d r o g e n a t e , w h i c h sho~vs t h e s ~ m e k i n e t i c s ~ as Fig_ 3 fi~r h y d r o g e n d e h y d r o g e n a s e a n d h a s a di~ulfidexlithiol g r o u p as an i n t e r m e d i a t e ~.n~. T h i s f l a v o p r o t e i n aL,~o c a t a l y z e s a n e x c h a n g e r e a c t i o n b e t w e e n I ) P N H anti d , : u t e r i m n o x i d e ~. S e v e r a l o f t h e flavop r o t e i n d e h y d m g e n a - , ~ s s h o w v a r y i n g e x c h a n g e r a t e s 2~.~2.m a n d in one c a s e C~ere ~s n o e x c h a n g e za. F l a v o p r o t e i n s a r e n o t u n i q u e in pr~)moting a n exchanT~'c• b e t w e e n a r e d u c e d s u b , i r a t e a n d w a t e r . T h e l|ytlrogena~e ~f D. des~tlfuricaits c a r r i e s o u t a n e x c h a r t g e b e t w e e n t h e e n z y m e h y d r i d e a n d d e u t e r i u m oxide, c~)ncurrently w i t h t h e r e v e r s a l n f a r e a c t i o n like E q n . ~. T h i s r e s u l t s in t h e a p p e a r a n c e o f t w o species o f h y d r o g e n i,~. D D a n d H D a n d t h e r e l a t i v e ratt. {)f fi~rmation o f D D t(~ H D is x.x { ~ e ref. 2}. T h e expe.'-iments w i t h t r i t i u m isot{)pe a n d h y d r o g e n d e h y d r o g e n a s e d o not distinguish between reacti~n x and the exchange between the enzyme and water. As t r i t i u m is i n c o r p o r a t e d r a p i d l y i n t o D P N H f r o m H T O , it c a n be c(~ncludeti f r o m t h e appeara~ace (ff l a b e l l i n g o n l y in the/q-l~)sition o f I ) P N H, t h a t t h e s t e r e o s p e c i f i c i t y o f t h e e x c h a n g e a n d dehydrogena.~., r e a c t i o n ~ arc t h e s a m e . T h e r e m o v a l (~f t h e lag p h 0 s e b y p r e i n c u b a t i n g t h e enzxa'~e w i t h 14: or D P N H c o u l d lm e x p l a i n e d b y t h e e x i s t e n c e o f t w o e ~ z y m i c t'orms, o n e being i n a c t i v e w h i c h is in e q u i l i b r i u m w i t h t h e a c t i v e f o r m . O n reductit,~a o f t h e a c t i v e f o r m , t h e i n a c t i v e f o r m is s l o w l y c o n v e r t e d i n t o t h e a c t i v e f o r m . Thi~ lag pha_ee c o n t r a s t s w i t h tit~,t d e s c r i b e d b y ~VIT'I'E?:BERC, AND R~.r,,~SKE ~s for e x t r a c t s o f H. eulropha w h i c h red u c e d I ) l ' Y w i t h H t a m i h a d a lag p h a s e t h a t wa:" n o t a f f e c t e d b y D P N H or Hz. T h e effect o f ~ulfide alsn d i s t i n g u i s h e s t h e I ) P N r e d , I c i n g .~.,stem:; in hi. eutr~pha a n d H. ruhla~tdii w h e r e in lht' f,~rmer sulfide a t i r r ~ l a t e ~ a c t i v i t y 3e a n d in t h e l a t t e r is a s t r o n g i n h i l , i t o r ( T a b l e II. ACKNO~VI,I:,I)t',E~II".N 1.'4
T h i s w o r k wa~ s u p p o r t e d b~ g r a n t s f r o m t h e N a t i o n a l I n s t i t u t e s ~,f H e a l t h a n d t h e N a t i o n a l .%cience Foundation;. T h e a u t h o r wishe~ to t h a n k Mr. A. ]~OSENBERG o f t h e C h e m i s t r y . D e p a r t m e n t . Y a l e U n i v e r s i t y f , r t h e a~..~ay o f t r i t i u m g a s i~t t h e e x c h a n g e experiment with tritiated water. f~!c, ch:m. 13~ophys÷ .4ct~. 67 (Iq(,3} 389 5q8
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