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8/13/2019 metaloenzym

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c o r r e s p o n d t o g r e a t d i f f er e n ce s in f o r m a l s y m m e t r y , a n d h e n c e a l so i nt h e a b s o r p t i o n s p e c tr a . T h e v a r i a ti o n s i n g e o m e t r y in d i c a t e d b y t h es p e c t ra l c h a n g e s o f C o ( I I ) c a r b o n i c a n h y d r a s e c o u l d th u s o c c u r w i t h o u tm u c h m o v e m e n t o f t h e l i ga n d s p ro v i d e d b y t h e p r o te in , b u t m i g h tp r i n c ip a l l y b e d u e t o t h e n u m b e r a n d e x a c t p o s i t i o n s o f l ig a n d s c o m i n gf r o m t h e s o l u t i o n e n v i r o n m e n t o f t h e a c t i v e s it e p o c k e t . P o s s ib l e e x p l a n a -t io n s w h y t h e a n i o n s m a y n o t b i n d i n a u n i f o r m m a n n e r w il l b e d i s cu s s edi n S e c t i o n I I I A 5 .

4 . T h e B o n d i n g o f C o ( I I ) t o t h e P r o t e i n

S i nc e t h e c y a n i d e - a n d c y a n a t e - c o m p l e x e s o f C o ( I I) c a r b o n i c a n h y d r a s e

a p p e a r t o a p p r o a c h t e t r a h e d r a l s y m m e t r y, a m o r e d e t a il e d a n a l y si s o ft h e i r a b s o r p t i o n s p e c t r a w a s a t t e m p t e d . F r o m t h e p o s i t io n s o f t il e c e n t e r so f t il e v is ib l e a n d n e a r - i n f r a r e d b a n d s , r e s p e c t i v e ly, a t e t r a h e d r a l l i g a n df ie ld sp l i tt in g , A t , o f a b o u t 5 . 3 k K w a s e s t i m a t e d5 8 ) . T h i s v a l u e i s q u i t el a rg e b u t c lo s e t o t h e t e t r a h e d r a l f ie ld s i n C o ( I I ) - b e n z i m i d a z o l e c o m -p l e x e s 6 2 ) . T h e m a g n e t i c m o m e n t o f t h e c y a n a t e c o m p l e x is i n a c c o r d -a n c e w i t h t h i s v a l u e o f / x t a s i l l u s t r a t e d i n Ta b l e 5 . To a c c o u n t f o r at e t r a h e d r a l f ie ld o f s u c h a s t r e n g t h i t m u s t b e a s s u m e d t h a t a t le a s ts o m e o f t h e t h r e e p r o t e i n l i g a n d s a r e b o n d i n g v i a n i t r o g e n a t o m s .

Table 5. Effec t i ve m agne t i c mom en t s [~eff)o f C o I I ) c a r b o n ic a n h y d r a s e 5 8 )

Com plex /~eff (B. M., ro omtempera ture)

Basic for m (OH -) 4.23 4- 0.10 aSulfanilamide 4.72 4- 0.04N CO - 4.41 4- 0.05 b

CI- 4.45 4- 0.08NO g 4.45 4- 0.03

a Pre liminary low -tempera ture measure-men t s P. A i s e n a n d S . L i n d s k o g ,un-published) ind icate a linear susceptibil-i ty increase with 1/T betw een 77 Kartd 2.3 K. H ence, eft is pra ctica llytempera ture- independent .

b A va lue of 4.30 B.M. is obtain ed aftercorrect ion for a small temperature-i l l -

dependen t pa ramagne t i sm wi th theassum pt ion of te t rahedra l coordinat ion12). Compare wi th [Co(I I ) -benzimida-

zole4] z+, /~eff= 4.28 B.M.62) after asimilar correction.

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U n f o r t u n a t e l y, t h e a c c u m u l a t e d p h y s i c a l a n d c h e m i c a l d a t a d o n o ty e t a l lo w t h e c o n c l u s iv e p o s i t i v e i d e n t if i c a ti o n o f t h e l i g a n d s , a l t h o u g hv a r i o u s c a n d id a t e s h a v e b e e n a d v a n c e d f r o m t i m e to t i m e 76,42, 63) .A m i n o g r o u p s a r e p r o b a b l y n o t i n v o l v e d in m e t a l b in d i ng , h o w e v e r , a st h e N - t e r m i n a l a - a m i n o g r o u p i s b l o c k e d w i t h a n a c e t y l g r o u p i n th en a t i v e e n z y m e 64), a n d t h e l y s y l r e s id u e s c a n b e c h e m i c a l l y m o d i f ie da t t h e e - a m i n o f u n c t i o n s w i t h o u t e f fe c t o n t h e c a t a l y t i c a c t i v i t y65, 66).T h e b o v i n e e n z y m e do e s n o t c o n t a i n a n y c y s t e in e 37,67) ,w h e r e a s b o t hh u m a n i s o e n z y m e s h a v e f f s in g l e S H - g r o u p . T h e s p e c t r a o f t h e c o r r e -s p o n d i n g Co II)e n z y m e s a r e v e r y s i m i l a r a n d i t w a s c o n c l u d e d38)t h a tm e t a l - b i n d i n g m u s t b e p r a c t i c a l l y i d e n t i c a l i n a l l t h e s e f o r m s , e x c l u d i n gs u l f u r a s a l i g a n d a l s o i n t h e h u m a n c a r b o n i c a n h y d r a s e s . T h i s w a sc o n f i rm e d i n th e X - r a y a n a l y s is o f t h e c r y s t a l s t r u c t u r e o f h u m a nc a r b o n ic a n h y d r a s e C . T h e d i s ta n c e b e t w e e n t h e S H - g r o u p a n d t h e z i n ci o n is a b o u t 1 4 A 6 1). H o w e v e r, t h e r e m u s t b e s o m e d i ff e re n c e s i n c o b a l tc o o r d i n a t i o n f o r t h e v a r i o u s f o r m s a s t h ed -d b a n d s o f t h e h u m a n c o b a lte n z y m e ]3 a t h i g h p H a p p e a r s t o b e o p t i c a ll y in a c t iv e i n c o n t r a s t t o t h eh i g h - s p e c i f i c a c t i v i t y f o r m s 39, 57, 68).T h e i n h i b i t o r c o m p l e x e s a r eo p t i c a l l y a c t i v e in a l l f o r m s F ig . 11). T h e o p t i c a l r o t a t o r y d i s p e r s i o na n d c i r c u l a r d i c h r o i s m o f c o b a l t c a r b o n i c a n h y d r a s e s h a v e b e e n f u l l yd i s c u s s e d b yCo lema n 51 , 68) .

S t r a ndbe rga n d c o w o r k e r s 69) h a v e c o ll ec t ed X - r a y d i ff r ac t io n d a t ao n th e h u m a n C e n z y m e t o 2.0 A r e s o lu t io n , a n d t h e c o r r e s p o n d i n gt h r e e - d i m e n s i o n a l e l e c t r o n d e n s i t y m a p h a s b e e n c o m p u t e d . A n a m i n oac id sequence de te rmina t ioni s i n p r o g r e s s 7 0 ) b u t t h e i n t e r p r e t a t i o n o ft h e c r y s t a l s t r u c t u r e i s a t a p r e l i m i n a r y s t a g e . T h e z i n c l i g a n d s h a v e n o tb e e n u n a m b i g u o u s l y i d en ti fi ed , b u t t w o o f t h e m b e a r t h e f e a t u r e s o fi m i d a z o l e r i n g s 69). T h e y o c c u r w i th i n t h e s a m e b r a n c h o f t h e p e p t i d ec h a i n i n a - H i s - X - H i s - s e q u e n c e . T h e t h i r d z i n c - p r o t e i n c o n t a c t i n-v o l v e s a d i ff e re n t p a r t o f t h e p r i m a r y s t r u c t u r e b u t i ts c h e m i c a l i d e n t i t y

is p r e s e n t l y u n k n o w n 1). T h e C o I I ) e n z y m e h a s n o t y e t b e e n s t u d ie d b yX - r a y m e t h o d s , b u t i ts c a t a l y t ic c a p a c i t y is a st r o n g i n d ic a t io n t h a t t h ed i ff e r en c e s i n t h e b i n d i n g o f t h e t w o m e t a l i o n s ar e s m a l l. F u r t h e r m o r e ,t h e s p e c t r a l p r o p e r t i e s o f t h e C o I I ) e n z y m e s e e m c o m p a t i b l e w i t h t h e c o-o r d i n a t io n g e o m e t r y o b s e r v e d f o r Z n I I ) i n h u m a n c a r b o n ic a n h y d r a s e C .

5 . T h e A n i o n B i n d i n g S i t e

T h e X - r a y a n a ly s is h a s a ls o b e e n e x t e n d e d t o i nc lu d e t h e i o d id e c o m p l e xo f h u m a n a n h y d r a s e C69, 7 1) . T h i s h e a v y a n i o n w a s f o u n d t o b e lo c a l iz e da t a p o s i t i o n v e r y c l o s e t o t h e m e t a l i o n a s h a d b e e n p r e d i c t e d o n t h e

1) Note added in proof: The third ligand has tentatively been identified as yetanother imidazole group.

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Fig . 11. Ci rcu lar d ichro ic spec t ra of Co I I ) carbonic a l lhydrase and complexes wi thsu l fonamides . A : ) bovine enzym e B; . . . ) bovine t3 enzym e p lus ace ta-

zo lamide , t3 : bovine 13 enzyme p lus ace tazolamide minus bovine 13 enzyme. C:) h u m a n B e n z ym e ; . . . ) h u m a n B e n zy m e p l u s e t h ox z o la m i d e . F r o mColeman 57)

b a s i s of t h e s p e c t r a l c h a n g e s o f t h e C o I I ) e n z y m e . T h e t e n t a t i v e z i nc -i o d i d e d i s t a n c e o f a b o u t 4 a p p e a r s t o b e s ig n i f i c a n t l y g r e a t e r t h a n t h a tf o r s m a l l c o m p l e x e s ca. 2 . 6 A ) , h o w e v e r . T i le a n i o n i s p o i n t i n g f r o m t h ez i n c i o n in t o t h e d e e p e r p a r t o f t h e a c t i v e - s i t e c r ev i ce . I t i s n o t y e t k n o w ni f t h e r e i s a b r i d g i n g w a t e r m o l e c u l e b e t w e e n i o d i d e a n d t h e m e t a l i o n

a s s u g g e s t e d b y P o c k e r a n d D i c k e r s o n 7 2 ) . T h e s e r e s u l t s s e e m t o b e a ru p o n t h e e a r l i e r o b s e r v a t i o n s t h a t t h e b i n d i n g s t r e n g t h s o f d i f f e re n ta n i o n s a r e to o g r e a t t o b e d u e t o a s i m p l e m e t a l - I i g a n d i n t e r a c t i o n i n a na q u e o u s m e d i u m . T h e f a c t t h a t t h e r e l a t iv e b i n d i n g s t r e n g t h s o f h a l i d e

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i on s a r e i n t h e o r d e r F - < C 1 - < B r - < I - ( Ta bl e 4 ) i n c o n t r a s t t o t h er eve r se o rd e r i n sma l l z i nc com plexes (57,73, 80),a lso sugges t t he i nvo lv e -m e n t o f a d d i t i o n a l f a c t o r s. M o r e o v e r, l a rg e a n i o n s s u c h a s N O ~ a n d

C 10 4, c o o r d i n a t i n g e x t r e m e l y lo o s e l y t o m e t a l i o n s i n w a t e r s o l u ti o n ,a r e t i g h t l y b o u n d i n t h e e n z y m e , w h e r e a s d i v a l e n t a n i o n s s u c h as S O ~ -d o n o t i n h i b it . T h e o b s e r v e d o r d e r o f r e l a ti v e e f fe c ts c o r r e s p o n d s r o u g h l yt o t h e l y o t r o p i c , o rHofmeister,se r i e s , wh ich i s r e l a t ed t o t he s i ze andh y d r a t i o n o f t h e a n i o n s a n d r e f le c ts t h e i r e f f ec t s u p o n t h e s t r u c t u r e o fw a t e r a r o u n d n o n - p o l a r g r o u p s7,1).

A n a n i o n - b i n d i n g s i te w i t h v e r y s im i l a r p r o p e r t i e s h a s b e e n d e m o n -s t r a t e d i n a n o n - m e t a l e n z y m e , a c e t o a c e t a t e d e c a r b o x y l a se f r o mClostri-diu m acetobutyricum 75). Fridovich 75)h a s d e t e r m i n e d t h e t h e r m o d y -n a m i c p a r a m e t e r s f o r a n i o n b i n d i n g i n t h i s e n z y m e . H e f o u n d a p r o p o r -t i o n a l i t y b e t w e e n A H a n d A S o f b i n d i n g . T h e r e is a n o p t i m a l a n i o n si zew h e r e b o t h t h e s e p a r a m e t e r s h a v e l a rg e n e g a t i v e v a l u e s . T h e i n t e r p r e t a -t i on o ff e r ed by F r id ov i ch l e ads t o a p i c tu r e o f t he an ion s i t e a s apositivelycharged holeo f a c e r t a i n s i z e w h i c h u p o n c h a rg e n e u t r a l i z a t i o n b e c o m e sm o r e h y d r o p h o b i c so t h a t a n e t o r d e r i n g o f t h e s u r r o u n d i n g w a t e r m o l e -cu les occurs .

T h e c o r r e s p o n d i n g t h e r m o d y n a m i c a n a l y s i s h a s n o t b e e n p e r f o r m e df o r c a r b o n i c a n h y d r a s e , b u t t h e p r e s e n c e i n t h e a c t i v e s i te o f a n o t h e r

p o s i t i v e l y c h a rg e d g r o u p h a s p r e v i o u s l y b e e n i n f e r r e d76, 77) .S u c ha n io n s as C N - o r S H - o r m i n g s t ro n g m e t a l b o n d s o f p a r t i a ll y c o v a l e n tc h a r a c t e r m i g h t b e b o u n d a t a m o r e " n o r m a l " d i s ta n c e t o th e m e t a l i o n,w h i c h p r e f e r s a s y m m e t r i c a l c o o r d i n a t i o n . T h e s e a n i o n s g i v e i n t e n s e l yc o l o r e d c o m p l e x e s w i t h t h e C o ( II ) e n z y m e as d i sc u s s ed in S e c t i o n I I I A 3 .O n t h e o t h e r h a n d , t h e m e t a l io n s ee m s to i n t e r a c t o n l y w e a k l y w i t ht h e l y o t r o p i c se ri es a ni o n s. T h e i r b i n d i n g m u s t b e c o n t r o l l e d b y t h eo v e r a l l e l e c t r o s t a t ic e n v i r o n m e n t i n t h e a n i o n s it e, s o t h a t t h e g e o m e t r yo f m e t a l - c o o r d i n a t i o n b e c o m e s i r r e g u l a r a n d s e n s i t i v e t o a n i o n s i z e .

T h e i r C o ( I I ) -e n z y m e co m p l e x e s a r e l es s i n t e n s e l y c o l o r e d a n d t h e s p e c t r aa r e b r o a d a n d v a r i a b l e (s ee S e c t i o n I I I A 3 a n d F i g . 9).

6 . T h e C a t a l y t i c M e c h a n i s m

U n t i l m o r e c o n c r e t e s t r u c t u r a l i n f o r m a t i o n is o b t a i n e d , t h e d i s c u s si o no n t h e c a t a l y t i c m e c h a n i s m o f c a r b o n i c a n h y d r a s e m u s t r e m a i n r a t h e rs p e c u l a ti v e . T h e e x p e r i m e n t a l e v i d e n c e re q u i r e s t h e p r e s e n c e in t h ea c t i v e s it e o f a b a si c g r o u p b e i n g i n s o m e m a n n e r l i n k e d t o t h e m e t a l i o n .

T h i s g r o u p i s g e n e r a ll y t h o u g h t t o p l a y a c r i ti c a l r o l e e it h e r a s a n u c l e o -p h i le i n a d i r e c t r e a c t i o n w i t h t h e s u b s t r a t e , o r t h r o u g h g e n e r a l b a s eca t l y s is . Sev e ra l s chem es fo rthe fun ctio n of carbonic anhydraseh a v e b e e np r o p o s e d 76, 47, 50, 78,79):

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a) I n t h e s i m p l e s t m o d e l , t h e p H - r a t e p r o fi le a n d t h e c o r r e s p o n d i n gs p e c t r a l c h a n g e o f t h e C o I I ) e n z y m e r e fl e c t t h e d i s s o c ia t io n a r o u n dp H 7 o f a p r o t o n f r o m a m e t a l - b o u n d w a t e r m o l ec u l ed T, 5 0 ) .T h e r e a c -t i o n o f t h i s O H - w i t h C 0 2 l e a d s t o a m e t a l - c o o r d i n a t e d H C O ~ . A l t er -n a t i v e ly, t h e O H - a c t in g a s a g en e r a l b a s e a c c e p t s a p r o t o n f r o m a w a t e rm o l e c u l e a t t a c k i n g C 0 2 . I n t h i s c a s e , a w a t e r - b r i d g e d m e t a l - H C O ~c o m p l e x i s f o r m e d .

I t h a s b e e n a r g u e d d 6 , 7 8 )t h a t t h e s e m e c h a n i s m s a r e l e s s l i k e l yb e c a u s e t h e p K a f o r t h e p r o c e ss ,

Me H20)~ + \ \ M e HsO )5O H+ +H +,

i s r a t h e r h i g h , a b o u t 9 f o r Z n 2+ o r C o 2+8 0 ) .H o w e v e r , th e h e x a q u o i on sa r e n o t r e p r e s e n t a t i v e m o d e l s fo r th e g e o m e t r i c a l a r r a n g e m e n t o f t h em e t a l i o n i n t h e e n z y m e o r f o r t h e e l e c t r o s t a t i c m i c r o e n v i r o n m e n t i nt h e a c t i v e s i t e . I t s e e m s r e a s o n a b l e t o a s s u m e t h a t t h e s a m e f e a t u r e s o ft h e a c t i v e s i te w h i c h s t a b i l iz e a n a n i o n - m e t a l i o n li n k a g e s ee p r e v i o u ss e c t i o n ) s h o u l d a l s o s t a b i l i z e a m e t a l - b o u n d O H - . H o w e v e r, t h e b a s i cs p e c t r u m o f t h e f r e e C o II ) e n z y m e is r a t h e r u n i q u e i n c o m p a r i s o n w i t ht h e s p e c t r a o b t a i n e d w i t h o t h e r a n i o n s , a n d t h i s h a s b e e n u s e d a s a na r g u m e n t f o r a l t e r n a t iv e m e c h a n i s m s J 6). O n th e o t h e r h a n d , O H -

d i f f e r s f r o m t h e o t h e r a n i o n s i n i t s c a p a c i t y t o p a r t i c i p a t e i n h y d r o g e nb o n d i n g .

S o m e o f t h e c u r r e n t i d e as o f t h e c a t a l y t i c s i te o f c a r b o n i c a n h y d r a s ea r e s u m m a r i z e d i n F i g . 1 2. A s d i s c u s s e d i n S e c t i o n I I I A 2 , l i tt l e is k n o w na b o u t t h e b i n d i n g o f C 0 2 . T h e p o s s i b il i ty o f a t r a n s i e n t m e t a l - c o o r d i -n a t e d C O 2 1 5 ) i s o p e n . N o e f f o rt h a s b e e n m a d e t o s k e t c h t h e d e t a i lso f t h e c a t a l y t i c m e c h a n i s m a s F i g. 1 2 u n d o u b t e d l y w i ll h a v e t o b er e v i s e d a f t e r t h e c o m p l e t i o n o f t h e c r y s t a l s t r u c t u r e d e t e r m i n a t i o n .

+

i

Fig. 12. Te nta tive scheme of the active site of carbonic anh ydrase.

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b ) A n o t h e r p o s s i b il it y w o u l d b e t h a t t h e m e t a l i on a n d a p r o t o nc o m p e t e f o r a p r o t e i n g r o u p i n t h e a c t i v e si te , so t h a t a n a d d i t i o n a la m i n o a c i d s id e c h a in b e c o m e s c o o r d i n a t e d a t h i g h p H16 , 46).To o b t a i na n a p p a r e n t p K ~ o f 7 f o r t h i s p r o ce s s, t h e s id e c h a i n m u s t , o f c o u r se ,b e m u c h m o r e b a s i c , w i t h a p K ~ ~> 1141 , 46, 50) .T h u s , h i s t i d i n e c a nb e e x c l u d e d a s s h o w n e x p l ic i tl y b yR i e p ea n d Wa n g 5 0 ) .O n t h e b i n d i n go f a n a n i o n , t h i s l i g a n d w o u l d b e d is p l a c e d a n d t a k e u p a p r o t o n . T h ec r y s t a l l o g r a p h i c e v i d e n c e d o e s n o t f a v o r t h i s a l t e r n a t i v e , h o w e v e r69).

c) T h e p r o t o n m i g h t i n s t e a d b e d e r i v e d f r o m a p r o t e i n g r o u p f u r t h e rr e m o v e d f r o m t h e m e t a l i o n b u t i n d i r e c tl y l in k e d t o i t, f o r e x a m p l e ,viah y d r o g e n b o n d s t o a c o o r d i n a t e d w a t e r m o l e c u l e (7 8), s o a s t o c o n t r o lt h e e x a c t g e o m e t r y o f t h e z i n c o r c o b a l t c o m p l e x . I t i s i m m e d i a t e l y c l e a r

t h a t t h i s s c h e m e m i g h t d i f fe r f r o m t h e o n e fi rs t d e s c ri b e d o n l y in t h ef o r m a l l o c a l iz a t io n o f c h a rg e s , a n d t h a t b o t h r e p r e s e n t e x t r e m e s o f o n egenera l case .

Pocke ta n d c o w o r k e rs 72, 78, 81, 82)h a v e p r e s e n t e d a g o o d d e a l o fa r g u m e n t a t i o n f o rhist idineb e i n g t h e a c t i v i t y - l i n k e d r e s id u e in a m e c h a -n i s m o f t h i s k i n d , a n d c l a im t h a t t h e p K ~ f o r t h e f o r m a t i o n o f m e t a l -b o u n d O H - is a b o v e 10 i n t h e b o v i n e e n z y m e . T h i s p r o p o s al is n o tc o n v i n c i n g , h o w e v e r, a s a n i m i d a z o l e g r o u p i n a b r i d g e d l i n k a g e t o t h em e t a l i on is n o t l ik e l y t o h a v e a n o r m a l p K a (s ee p a r a g r a p h b ) . F u r t h e r -

m o r e , a d i f f e r e n t s o u r c e fo r t h e 1 -- 2 p r o t o n s d i s s o c i at e d o n t h e b i n d i n go f z in c to t h e a p o p r o t e i n a t p H 837 , d3)m u s t b e p r e s en t . F u r t h e rs t u d i e s o f t h e C o ( I I) e n z y m e m i g h t b e h e l p f u l i n f in d i n g a l t e r n a t i v ee x p l a n a t i o n s f o r t h e o b s e r v e d i n c r e a s e i n e s t e r a s e a c t i v i t y a t v e r y h i g hp H (78, 82). I t i s a l r e a d y a p p a r e n t f r o m t h e s p e c t r a s h o w n i n F i g . 4t h a t n o d r a s ti c c h a n g e i n m e t a l - c o o r d in a t i o n h a p p e n s b e t w e e n p H 7 .8and 11.6.

Wa n g (79) ha s p rop ose d a som ew ha t d i f f e r en t r o l e fo r an ac t i ve s i t eh i s t i d i n e r e s i d u e , i n v o l v i n g t h e p r o m o t i o n o f aproton t ransferf r o m am e t a l - b o u n d O H - t o a n o x y g e n a t o m o f C O 2 (cf. F i g. 12) s o t h a t a l a n ef r a c t i o n o f t h e n e g a t i v e c h a rg e o f t h e H C O ~ b e c o m e s lo c a l iz e d to t h e 0a t o m i n c lo s e st c o n t a c t w i t h t h e p o s i t i v e l y c h a rg e d m e t a l i on . H o w e v e r,i n v i e w o f t h e p r e s e n t c o n c e p t o f t h e b i n d i n g o f t h i s t y p e o f a n i o n s (s eet h e p r e v i o u s s ec ti o n ) su c h a p r o t o n t r a n s f e r m a y n o t b e a n e c e s s a r y s te pi n t h e c a t a l y t i c r e a c t i o n .

T h e p r e s e n c e o fimidazole groupsi n t h e a c t i v e s i te r eg i o n o f h u m a nc a r b o n i c a n h y d r a s e B h a s , i n f a c t , b e e n d e m o n s t r a t e d b y c h e m i c a lm o d i f ic a t io n . T h u s , b r o m o a c e t a t e r e a c t s s p e c i fi c al ly w i t h t h e 3 ' - N o f a

h i s ti d in e r e s id u e t o g iv e a p a r t ia l l y a c t i v e m o n o c a r b o x y m e t h y l e n z y m e65).T h e r e a c ti o n d e p e n d s o n t h e i n it ia l c o m b i n a t i o n o f t h e b r o m o a c e t a t e

i o n w i t h t h e a n i o n b i n d i n g s i t e65 , 83).I n a d e t a i l e d s t u d y,Bradbury 83)h a s s h o w n t h a t t h e i rr e v e r s ib l e r e a c t i o n a t s a t u r a t i o n w i t h i o d o a c e t a t e

1 2 S t z u c t ur e a n d B o n d i n g ~ Vo l 8 7 7


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requires the basic form of a group in the enzyme-inhibitor complexhaving a p K a about 5.8. This p K a should represent the reactive histidinewhich is thus neither identical with the activity-linked group of pK~ 7

nor likely to be a zinc ligand. The correspondingly modified co E)enzyme has a spectrum of the same type as other anion complexes.There is a ~bH-dependent spectral change, but it is shifted to higher pHcompared to the unmodified enzyme. The pH-ra te profile of the residualac tiv ity of the modified enz yme is similarly shifted (Fig. 13). Theseresults are simplest rationalized in terms of the covalent fixation of ananionic inhibitor.

I I ~

I00 M ~Z n ( E ) o n d C o ( ~ ) - E N Z Y

8O~ / / o I I

~ /22kd

W

20 /f CM ENZYMES

7 /gx

0 ( I ~ , I , q ,

7 8 9pH

Fig. 13. The pH dependence of the esterase activity of ative and carboxymethyl-ated human carbonic anhydrase ]3. The term C2i-enzymes refers to Zn(II)-enzyme0 , &) and Co(II)-enzyme (O, A) which had been carboxymethylated with bromo-

acetate. Relative activity was estimated in each experiment by taking 100% as thevalue corresponding to maximal activity of the same enzyme at high pH. Thisvalue was 17 min -1 for the unreacted enzyme and 3.5 and 2.1 rain -1 for the ZIx(II)-and Co(II)-carboxymethylated enzymes, respectively. From W hitn ey et al. 65)

A second histidine residue can be modified at the 3'-position inhum an carbonic anhydrase B with a N-chloroacetyl sulfonamide 84, 85)

yielding an inactive product.The mere fact that imidazole is present near the metal ion suggests

that it should have some function, but the data do not allow any uniqueconclusion about its possible role in catalysis. Very probably there are

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structural differences between the active site regions of the human 13form and the high specific activity forms 53) as tile latter enzymes arenot modified by the same reagents. Furthermore, preliminary sequence

studies on the human C form suggest that different residues are presentin this enzyme at the positions of the modifiable histidines in the sequenceof the B form (70). Specific modifications of histidine residues leadingto act ivi ty losses have been achieved for the human C form with bromo-pyruvate (86) and for the bovine enzyme with bromoacetazolamide 87),however.

The chemical modification studies have thus not yet led to a muchmore conclusive picture of the active site than that outlined in Fig. 12,and the identification of a m i n o a c i d s i d e c h a i n sinvolved in catalysis or

substrate binding may have to await the completion of the crystalstructure determination. The reporter properties of the Co(II) enzymeclearly show, however, th at an open coordination position is of decisivefunctional importance, that the metal ion is intimately associated withthe basic group participating in the reaction, and that the metal ion isprobably also involved in the binding of one of the substrates, HCO~.

B. Carboxypeptidase

Carboxypeptidases A and 13 are formed by the hydrolyt ic action of trypsinupon inactive precursors, procarboxypeptidases. These zymogens aresynthesized in the pancreas from which they can be isolated 88). Depend-ing on the preparation method, different forms of carboxypeptidase Aare produced, varying in the N-terminal region 89).

As indicated by their name, these enzymes are exopeptidases catalyz-ing the c leavage o f t he C- t e rm ina l pep t ide bondof the substrate molecule.

The specificities of the A and B enzymes differ markedly in that theformer prefers an aromatic of hydrophobic aliphatic side chain in the C-terminal residue of the substrate, whereas the latter requires a positivelycharged side chain in the same position 88). Typical low-molecularweight substrates are carbobenzoxyglycyl-L-phenylalanine and hippurylL-arginine for the A and B enzymes, respectively. The carboxypeptidasesalso catalyze the hydrolysis of certain esters, such as hippuryl-L-fl-phenyllactic acid and hippuryl-L-arginic acid, respectively. Dipeptideshaving unblocked e-NH2 groups are hydrolyzed only very slowly.

Bovine carboxypeptidase A has a molecular weight of 34,000 and iscomposed of a s i n g l e p e p t i d e c h a i n .Carboxypeptidase B has similarproperties and is probably a homologous protein 90). Both enzymescontain one z inc ion pe r molecu le .Carboxypeptidase A has been exten-

12. 179


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S . L i n d s k o g

s i v e ly s t u d ie d , m a i n l y i n t h e l a b o r a t o ri e s o fH . N e u r a t ha n d B. L . Va l lee ,b u t t h e p h y s i c a l p r o p e r ti e s o f t h eCo II)e n z y m e h a v e a s y e t n o t b e e nf u l l y u t il iz e d . M o s t o f t h e k n o w l e d g e o f t h e f u n c t i o n o f t h e e n z y m e r e s t son o the r k inds o f ev idence . However, i t f a l l s ou t s ide the scope o f t h i sr ev iew to g ive a fu l l accoun t o f t ha t a spec t .

T h e d e t e r m i n a t i o n o f t h e c r y s t a l s t r u c t u r e o f c a r b o x y p e p t i d a s e Aa n d i t s c o m p l e x w i t h g l y c y l- L - t y r o si n e h a s b e e n c o m p l e t e d t o 2 . 0 Ar e s o lu t io n b yL i p s c o m ban d coworkes 91 ) . A de t a i l ed desc r ip t ion o f t hem o l e c u l e i n c l u d i n g a c o m p r e h e n s i v e d i s c u s s i o n o f s t r u c t u r e - f u n c t i o nr e l a ti o n s h a v e b e e n p r e s e n t e d91).

1. M e t a l I o n S p e c i fi c it y a n d S u b s t r a t e B i n d i n gC a r b o x y p e p t i d a s e A w a s t h e f i rs t m e t a l l o e n z y m e w h e r e t h e f u n c t i o n a lr e q u i r e m e n t o f z i n c w a s c l e a r l y d e m o n s t r a t e d9, 92).I n s i m i l a r i t y t oc a r b o n ic a n h y d r a s e , th e c h e l a t in g s i te c a n c o m b i n e w i t h a v a r i e t y o f m e t a lions 93), b u t t h e a c t i v a t i o n s p e c if ic i ty i s b r o a d e r. S o m e m e t a l i o n s ,Pb 2+, Cd u+ an d H g 2+, y i e ld o n ly e s t e r a se ac t iv i ty b u t f a i l t o r e s to re t hepep t idase ac t iv i ty. Of a va r i e ty o f ca t ions t e s t ed , on ly Cu 2+ g ives ac o m p l e t e l y i n a c t i v e e n z y m e . I n t h e s t a n d a r d p e p t i d a s e a s s a y, c o b a l te a r b o x y p e p t i d a s e i s t h e m o s t a c t i v e m e t a l d e r iv a t i v e , w h i l e i t h a s a b o u tt h e s a m e e s te r a se a c t i v i t y a s t h e n a t i v e e n z y m e93 , 94) ,Table 6) .K i n e t i c a l l y, t h e C o I I) e n z y m e s ho w s t h e s a m e q u a l i t a t i v e f e a t u r e s a st h e n a t i v e e n z y m e95),a n d t h e q u a n t i t a t i v e d i f fe r en c e s a r e n o t r e s t r ic t e dto a s ing le k ine t i c pa ra m e te r.

Ta b l e 6 . M e t a l i o n s p e c i f ic i t y o f c a r b o x y p e p t i d a s e A a

M e t a l i o n P e p t i d a s e a c t i v i t yC a r b o b e n z o x y g l y c y l -L - p h e n y l a l a n i n e

E s t e r a s e a c t i v i t yH i p p u r y l - d l - f l-p h e n y l l a c t a t e

Z n 2 7 . 5 1 . 1 5C o 2 1 2 . 0 1 . 1 0

N i ~ 8 . 0 1 . 0 0M n 2 0 , 6 0 . 4 0C u 2 0 0H g 2 0 1 .3 4C d 2 0 1 . 7 5P b 2 0 0 . 6 0

a F r o m C o l e m a na n d Val l ee 93 ) .S e e t h i s p a p e r f o r d e f i -n i t i o n o f u n i t s a n d f o r a s s a y c o n d i t io n s .

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T h e s u b s t r a t e c o n c e n t r a t i o n d e p e n d e n c e o f t h e c o b a l t c a r b o x y -p e p t i d a s e B r e a c t io n s h a s a ls o b e e n s t u d i e d96).W i t h m o s t o f t h e i n v e s-t i g a t e d s u b s t r a te s , t h e h i g h e r a c t i v i t y o f t h e c o b a l t e n z y m e a s c o m p a r e dto the z inc enzyme i s e s sen t i a l l y exp res sed in l a rge r va lues o f Vmax ,whi le Kin-va lues a re s imi la r.

In t he p re sence o f a la rge excess o f Co 2+ , bo th n a t iv e 97 ) an d coba l t9 2 ) c a r b o x y p e p t i d a s e A s h o w a n a p p r o x i m a t e l y t w o - f o l d a c t i v i t y

i n c re a s e. T h e k i n e t i c s of t h e e n z y m e a r e v e r y c o m p l e x a t m o d e r a t e o rh i g h s u b s tr a t e c o n c e n t r a ti o n s a n d i n v o lv e b o t h a p p a r e n t a c t i v a t io n a n di n h i b i t i o n b y s u b s t r a t e 95). U n d e r t h e s t a n d a r d a s s a y c o n d i t i o n s u s e di n c o n n e c t i o n w i t h t h e o b s e r v e d c o b a l t a c t i v a t i o n , a l l t h e s e c o m p l i c a ti n gfac to r s con t r ibu te s ign i f ican t ly. Th e add i t i ona l Co 2+ poss ib ly in t e r f e re s

w i t h th e s e s e c o n d a r y e f f ec t s r a t h e r t h a n b e i n g a p a r t i c i p a n t i n c a t a ly s i s.F u r t h e r e x p e r i m e n t a t i o n i s n e e d e d t o c l a r i f y t h i s d e t ai l.

P e p t i d e s u b s tr a t e s h a v e be e n sh o w n to b i n d t o t h e a p o e n z y m e a n dp r o t e c t i t f r o m r e a c t i v a t i o n w i t h m e t a l i o n s98, 99).T h e a p o e n z y m e -s u b s t r a t e c o m p l e x e s w e r e e s t i m a t e d t o h a v e a b o u t t h e s a m e st a b il it ie s a st h e c o r r e s p o n d i n g co m p l e x e s w i t h t h e n a t i v e e n z y m e . O n t h e o t h e rh a n d , e s t e r s u b s t r a t e s a p p e a r t o r e q u i r e t h e p r e s e n ce o f t h e m e t a l i o nf o r b i n d i n g . M e t a l lo c a r b o x y p e p t i d a s e s , i n c l u d in g t h e i n a c t i v e C n I I )e n z y m e , f o r m c o m p l e x e s w i t h b o t h k i n d s o f s u b s t r a t e s h i n d e r i n g t h e

d i s soc i a t ion o f the m e ta l i on .A c e t y l a t i o n a n d i o d i n a t i o n o f d i s c r e t e t y r o s y l r e s i d u e s h a v e b e e n

a c h i e v e d , l e a d i n g t o a n a b o l i s h m e n t o f p e p t i d a s e a c t i v i t y a n d a n e n h a n c e -m e n t o f e s te r a s e a c t i v i t y f o r b o t h n a t i v e a n d c o b a l t c a r b o x y p e p t i d a s e A7 0 0 ) .

2 . M e t a l I o n B o n d i n g

T h e 2 . 0 / ~ e le c tr o n d e n s i t y m a p o f c a r b o x y p e p t i d a s e A s h o w sthree z inc-

pr ote in contacts 91) .The l i gands have been iden t i f i ed a s h i s t i d ine -69 ,g lu t a m ic ac id -72 and h i s t i d ine -19697, 707),w h e r e t h e n u m b e r s i n d i c a tet h e p o s i ti o n s o f t h e r e si d u es i n t h e s e q u e n c e c o u n t e d f r o m t h e N - t e r m i n a le n d . T h e g e o m e t r y o f t h e c o m p l e x i s i r re g u l a r b u t r e se m b l e s a d i s t o r t e dt e t r a h e d r o n w i t h a n o p e n p o s i t io n d i r e c t e d t o w a r d s t h e a c t i v e s i te p o c k e t ,a n d p r e s u m a b l y o c c up ie d b y w a t e r in t h e r e s ti n g e n z y m e 9 1 ). T h es i m i l a r i t y w i t h t h e t e n t a t i v e s t r u c t u r e o f t h e m e t a l - b i n d i n g s it e i nc a r b o n i c a n h y d r a s e i s s tr i k in g .

W h i l e t h e c r y s t a l s t r u c t u r e c o n f i r m s s e v e r a l i n f e r e n c e s a b o u t t h e

c a t a l y t i c s i te b a s e d o n c h e m i c a l s t u d i e s o f t h e e n z y m e i n s o l u t io n 1 00 ,702),p r e d i c ti o n s a s t o m e t a l b o n d i n g16, 703)were less successfu l . Thecoo rd ina t ion o f z inc o r coba l t) t o two p ro t e in l i gands , an -SH a nd thet e r m i n a l ~ - N H 2 , h a d b e e n p o s t u l a t e d p r i n c i p a l l y f r o m s t u d i e s i n v o l v i n g

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g r o u p - s p e c i fi c r e a g e n t s , d e t e r m i n a t i o n s o f s t a b il it ie s o f m e t a l - c a r b o x y -p e p t i d a s e c o m p l e x e s , a n d t h e a b s o r p t i o n s p e c t r u m o f t h e c o b a l t e n z y m e .I t h a d b e e n p o i n t e d o u t 38, 704) , h o w e v e r , t h a t t h e s e e x p e r i m e n t s

p r o v i d e d n o c o n c l u s i v e d e m o n s t r a t i o n o f S , N - c o o r d i n a t io n . F o r e x a m p l e ,t h e r e l a ti v e m a g n i t u d e s o f t h e s t a b i l i ty c o n s t a n t s g i v e n in Ta b l e 7 a r e

Table 7. Apparent stability constants for metal binding in procarboxy-peptidase A, carboxypeptidase A and human carbonic anhydrase B

Metal ion log Kapp

proca rbox y- carbo xy- humar~ carbonicpept idase A a pept idase A a anh ydras e B b

Mn2+ 3.4 4.6 3.8Co2+ 5.4 7.0 7.2N i 2+ 5.9 8.2 9.5Cu 2+ 8.1 10.6 11.6Zn 2+ 9.0 10.5 10.5Cd 2+ 8.4 10.8 9.2H g 2+ 18.3 21.0 21.5

a F r o m Pi ras a n d Vallee 107) ; p H 8; 4 C.b F r o m Lindskogand N y m a n 3 8 ); p it 5.5 ; 23 C.

v e r y s i m i l ar t o t ile c o r r e s p o n d i n g v a l u e s f o r c a r b o n i c a n h y d r a s e h a v i n gn e i t h e r a t h io l g r o u p n o r a n a - a m i n o g r o u p a s m e t a l l ig a n d s. A l t h o u g ht h e a b s o l u te m a g n i t u d e s o f t h e s t a b i l i ty c o n s t a n t s , K a pp , c o m p a r e c l o se l yt o K 1 f o r s o m e b i d e n t a t e l ig a n d s , it w a s n o t c o n s i d e r e d t h a t t h e m e t a l -b i n d i n g t o t h e p r o t e i n i n v o l v e s th e d i s p l a c e m e n t o f a p r o t o n a t t h e p H

o f m e a s u r e m e n t 93) s o t h a t K a pp m u s t b e a f u n c t io n o f p H . T h e r e s u l tso f t h e m e a s u r e m e n t s o f t h e r a t e s a n d h e a t o f t h e r e a c t i o n b e t w e e n Z n 2+a n d a p o c a r b o n i c a n h y d r a s e 4 2, 4 3) d i s c u s s e d i n S e c t i o n I I I A1 a m p l yi ll u s tr a te p r o b l e m s i n h e r e n t in c o m p a r i n g th e r m o d y n a m i c p r o p e r t i e s o fm e t a l - b i n d i n g t o s p e ci fi c p r o t e i n s it e s a n d t o s m a l l li g a n d s i n w a t e rs o l u t i o n .

T i le v is ib le a b s o r p t i o n s p e c t r u m o f c o b a l t c a r b o x y p e p t i d a s e A w a sa t a n e a r l y s t a g e t h o u g h t t o b e c h a r a c t e r i s t ic o f s u l f u r c o o r d i n a t i o n 1 6,705) . T h e o r i g in a l ly p u b l is h e d s p e c t r u m94) i s p o o r l y r e s o l v e d F i g . 14 ),

b u t a m o r e d e t a il e d s t u d y h a s r e c e n t l y b e e n a n n o u n c e d J 06 ). M a x i m aa t 5 5 5 n m e = 1 60 M - 1 c m - 1 ) a n d 5 7 2 n m e = 1 60 M - 1 c m - 1 ) , a s h o u l d e ra t 5 0 0 n m a n d a n e a r - i n f ra r e d b a n d c e n t e r e d a t 9 4 0 n m e = 2 5 M - 1 c m - 1 )a r e r e p o r t e d . T h e r e s e e m s t o b e a c le a r r e s e m b l a n c e w i t h s p e c t r a l f o r m s

8 2


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Co ba l t I I ) i n M eta lloenzymes

of cobalt carbonic anhydrase, particularly the chloride complex Fig. 9).The spectral features seem compatible with the geometry and bondingobserved for Zn ~+ in the crystal structure.

.751 I I

o

.50

._>

\ cP 0 col2 5t

0.0

3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0

X i n r n p

Fig. 14. Absorption spectra of Co II)-carboxypeptidase A, [ CP D)C o], na tiveca rboxypep t idase A , [ CPD)Zn] , a ixd Co H20)~+, respect ive ly. FromColeman alXdValle 94)

Metal binding in ibrocarboxypeptidase is weaker than in the activeenzyme 707), Table 7). It was proposed th at the bonding involvessulfur and a weaker ligand than N 507). In view of the present conceptof the chelating site in carboxypeptidase, further studies of the zymogenare necessary. In tha t connection, the cobalt complex should be valuable.

3. The Catalytic Mechanism

The binding of glycyl-L-tyrosine n the active site pocket of carboxypep-tidase A is illustrated in Fig. 15. Tyrosine-248 and glutamic acid-270

are believed to participate in the catalytic reaction and represent theacidic and basic groups, respectively, involved in the bell-shaped pH-rate profile. In the bond-cleavage reaction, the carboxyl group of Glu-270may act by a nucleophilic attack on the carbonyl group while Tyr-248

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S. Lindskog

d o n a t e s a p r o t o n t o t h e a m i d e n i t r o g e n o f t h e s u s c e p t i b le p e p t i d e b o n d .T h e li n k b e t w e e n G l u -2 7 0 a n d t h e ~ - - N H 2 o f t h e d i p e p t i d e e x p l ai n s w h yt h i s s u b s t r a t e i s n o t e f f ic i e n t ly h y d r o l y z e d . F o r a d e t a i l e d d i s cu s s io no f t h e s t r u c t u r e a n d p o s s ib l e c a t a l y t i c m e c h a n i s m s , t il e r e a d e r is r e f e rr e dt o t h e e x c e ll e n t p a p e r b yL@ scom b et al . 91),w h e r e a ls o t h e i n t e r e s t i n gs u b s t r a t e - i n d u c e d c o n f o r m a t i o n a l c h a n g e s a re d e s c ri b e d .

1+145

H i s - - Z n - - H s ~H , ~,1 9 6 I ' 6 9 , ' . . . . n u r y r 2 4 8

G l u 7 2 0 ~

C H 2

G t u - . H 2 N2 7 0 .

Fig. 15. Schematic drawing of the active site region of carboxypeptidase A intera ct-ing with glycyl-L-tyrosine.FromLipscomb el al.(97) aad sequence information fromthe laboratoryof H . Neurath 707).

T h e probable func t ion of the meta l ioni n c lu d e s t h e b i n d i n g a n d p o l a r i-z a t i o n o f t h e c a r b o n y l g r o u p o f t h e s u s c e p t i b l e p e p t i d e b o n d . I n t h eg l y c y l - L - t y r o s in e c o m p l e x , t h e m e t a l i o n is s h i ft e d a b o u t 0 . 5 / ~ t o w a r d st h e s u b s t r a t e r e l a t i v e to i t s p o s i t i o n i n ti le r e s t i n g e n z y m e ( g J) . F u r t h e r -m o r e , Late a n d Vallee 106)h a v e r e p o r t e d t h a t t h e s p e c t r u m o fCo II)c a r b o x y p e p t i d a s e is c h a n g e d o n t h e b i n d i n g o f t h i s s u b s t r a t e . T h i s

o b s e r v a t i o n s h o u ld o p e n n e w p o s si b il it ie s o f t e s t i n g a l t e r n a t i v e m e c h a -n i s m s o n t h e b a s is o f t h e c r y s t a l s tr u c t u r e , a n d d e t e c t i n g t r a n s i e n t i n t e r -m e d i a t e s d u r i n g t h e c a t a l y t ic i n t e r a c ti o n o f c a r b o x y p e p t i d a s e w i t h i tsr a p i d l y h y d r o l y z e d s u b s t r a te s .

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Cobalt II) in Metalloenzymes

C Alkaline Phosphatase

A l k a l i n e p h o s p h a t a s e s f o r m a w i d e s p r e a d g r o u p o f r e l a t i v e l y u n s p e c if ice n z y m e s c a t al y z i n g th e h y d r o l y s i s o f m a n y o r t h o p h o s p h a t e m o n o e s t e rs .T h e i r p H o p t i m a a r e g e n e r a l l y a t p H 8 o r a b o v e . S e v e r a l a l k a li n e p h o s -p h a t a s e s h a v e b e e n s h o w n t o c o n t a i n z i n c 3 ).

T h e e n z y m e i s o la t ed f r o m th e b a c t e r i u mEscher ich ia co l ih a s b e e ne x t e n s i v e l y c h a r a c t e ri z e d . I t s m o l e c u l a r w e i g h t is a b o u t 8 0 , 0 00108,109 .I t c a n b e d i s s o c i a t e d i n t o h a l f - m o l e c u l e s b y a c i d t r e a t m e n t108).R e a s s o c ia t io n c a n b e b r o u g h t a b o u t a t n e u t r a l p H . Z i n c i on s p r o m o t eth i s p roces s 108) ,b u t d i m e r i z a ti o n c a n o c c u r i n t h e a b s e n ce o f m e t a li on s a t p r o t e i n c o n c e n t r a t io n s l a rg e r t h a n 1 m g / m l109, 110).On d i a ly s i so f t h e e n z y m e ag a i n s t c h e l a t i n g a g e n t s a d i m e r ic , in a c t i v e a p o p r o t e i ni s p r o d u c e d , s h o w i n g t h a t t h e m e t a l i o n is a ls o r e q u i r e d f o r a c t i v i t y 111 ).T h e b i n d i n g o f Z n 2 + t o t h e a p o e n z y m e i n v o l v e s s o m e a l t e r a t i o n s i nc o n f o r m a t i o n a s i n d i c a t e d b y c h a n g e s i n o p t i c a l r o t a t i o n a n d u l t r a v i o l e ta b s o r p t i o n s p e c t r u m 110).

T h e h y d r o l y s i s r e a c t io n i s t h o u g h t t o p r o c e e dv ia a c o v a l e n t e n z y m e -p h o s p h a t e i n t e r m e d i a t e112) :

E + s x----x ES ) EP1 + RO H ~ E + Pt + RO H

w h e r e P 1 r e p r e s e n ts o r t h o p h o s p h a t e a n d R 0 H t h e a l co h o li c m o i e t y oft h e e s t e r, S . T h e r a t e o f d e p h o s p h o r y l a t i o n is li n k e d t o t h e i o n i z a t i o n o fa g ro u p o f p K a b o u t 7 .5173)s o t h a t t h e p h o s p h o r y l a t i o n re a c t io n b e -c o m e s r a te - l im i t i n g a t h i g h p H . A t l o w p H , t h e i n t e r m e d i a t e is o n l ys l ow l y h y d r o l y z e d , a n d a m o n o p h o s p h o r y l - e n z y m e c a n b e i so l at e d,s u g g e s t in g t il e p r e s e n c e o f o n e a c t i v e s i te p e r d i m e r. T h e p h o s p h a t e g r o u pi s e st e ri f ie d t o t h e h y d r o x y l g r o u p o f a s e ri n e re s i d u e112). H o w e v e r ,t h e p o s s i b i li t y o f t w o i n t e r d e p e n d e n t a c t i v e s it e s s h o u l d b e k e p t i n m i n dcf. S e c t i o n I I I c 2 .

1. M e t a l I o n B i n d i n g

T h e d im e r i c n a t u r e o f a l k a l in e p h o s p h a t a s e m a k e s i t a m o r e c o m p l i c a t -e d s y s t e m t h a n c a r b o n i c a n h y d r a s e o r c a r b o x y p e p t i d a s e . T h e e n z y m ec o n t a i n s s e v e r a l m e t a l - b i n d i n g s i t e s . T h e s t o i c h i o m e t r y o f z i n c b i n d i n gis n o t c o m p l e t e l y s e tt le d . T h e r e a r e a t l e a s t t w o s t r o n g l y b o u n d m e t a li ons 109, 111, 114),b u t t he p r e sence o f f ou r spec if ic s i t e s ha s beenc l a i m e d 715, 116).A t a lk a li n e p H , t h e e n z y m e t e n d s t o b i n d e v e n m o r e

z in c r a t h e r s t r o n g l y, b u t p r o b a b l y t o s i te s u n r e l a t e d t o c a t a l y t ic f u n c t i o n109).A c r i t i c a l eva lua t i on o f t h i s a spec t f a l l s ou t s ide t he s cope o f t h i s

r e v ie w, b u t i t a p p e a r s t h a t s o m e o f t il e a p p a r e n t d i s c re p a n c i es a r e d u et o d i ff e re n t e x p e r i m e n t a l m e t h o d s i n m e a s u r in g m e t a l b i nd i n g .

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S. Lilldskog

T h e p u b l i s h e d v a l u e s o f t h ebinding strength o f Z n 2+s h o w a n e v e nw i d e r v a r i a t i o n 115--118).O n l y i n o ne c a se h as a n a t t e m p t b e e n m a d et o m e a s u r e t h e b i n d i n g d i r e c t l y b y e q u i li b r iu m d i a ly s i s w i t h 6 5Z n a n dc o m p e t i n g c h e l a ti n g a g e n t s ( 11 7). T h i s s t u d y i n d i c a t e d t h a t t w o s t r o n gs i t e s a r e e q u i v a l e n t a n d i n d e p e n d e n t . A s i m i l a r s t u d y, w h e r e b i n d i n gw a s e s t i m a t e d b y m e a s u r i n g e n z y m i c a c t iv i t y, su g g e s t e d a w e a k e r b i n d -ing fo r t he second z inc ion118).

T h e m e t a l l i g a n d s a r e n o t k n o w n , b u t i m i d a z o l e h a s b e e n p r o p o s e df r o m t i t r a t i o n s t u d i e s 11d) an d o n the bas i s o f t he p ro t ec t ive e ff ec t o fz i n c a g a i n s t m o d i f ic a t i o n o f h is t id i n e b y p h o t o o x i d a t i o n119).

I n s p i t e o f t h e d i s a g r e e m e n t i n t h e l i t e r a t u r e a b o u t t h e n u m b e r o fi n t r i n s i c z i n c i o n s i n a l k a l i n e p h o s p h a t a s e , m o s t d a t a s e e m t o c o n c u rt h a t o n l y t w o s i te s a r e l in k e d t o t h e c a t a l y t i c f u n c t i o n , w h i l e a d d i t io n a lb i n d i n g m a y h a v e m a rg i n a l e f f e c t scf.,however, re f .110).

2 . T h e C o b a l t( I I) E n z y m e

Z i n c ( II ) a n d C o ( II ) a r e t h e o n l y c a ti o n s f o u n d t o r e a c t i v a t e a p o p h o s -p h a t a s e t o a n y a p p r e c i a b l e e x t e n t120).T h e Co II)e n z y m e f o l lo w s t h es a m e f o r m a l m e c h a n i s m a s t h e n a t i v e e n z y m e , b u t h a s a lo w e r s pe c if ica c t i v i t y 113, 721) .I t l a c k s t h e p h o s p h o t r a n s f e r a s e a c t i v i t y113 , 119,121) obse rved fo r t he na t ive enzyme , fo r example in Tr i s bu ffe r s . Th i sw a s t a k e n t o i m p l y t h a t t h e l o w e r a c t i v i t y o f t h e c o b a lt e n z y m e is d u et o a l o w e r r a t e o f p h o s p h o r y l a t i o n , so t h a t t h i s s t e p b e c o m e s r a t e - l im i t i n ga lso be lowp H 7 113).S t o p p e d -f lo w e x p e r i m e n t s b yGo ttesman et al. 121)s h o w, h o w e v e r, t h a t a v e r y f a s t b u r s t o f p - n i t r o p h e n o l oc c u rs i n t h ec o b a l t a l k a li n e p h o s p h a t a s e - c a t a l y z e d h y d r o l y s i s o f p - n i t r o p h e n y l p h o s -p h a t e o v e r a w i d e p H r e gi on . T h e s e r e su l ts s t r o n g l y s u g g e s t t h a t a s t e ps u b s e q u e n t t o t h e p h o s p h o r y l a t i o n is ra t e - l im i t i n g i n t h i s m e t a l d e r i v a t i v e .

Co 2+ i s le s s s t rong ly bo un d to a lka l ine pho sph a ta se th an Zn 2+109,116, 122).T h e v e r y s m a l l s t a b i l i t y c o n s t a n t (lo g K ~ 4 .1 ) r e p o r t e d b yLa zdu nski et al. 115)m u s t b e c o n s id e r e d u n r e p r e s e n t a t i v e f o r t h e f u n c -t iona l i ons , however.

R e c e n t l y, t w o p a p e r s h a v e a p p e a r e d r e p o r t i n g t h e s p e c t r a l p r o p e r ti e so f coba l t a lka l ine phospha ta se . Accord ing toSimpsona n d Vallee 115)add i t i on o f two e qu iva l en t s o f Co 2+ to t h e apo enzy m e y ie lds on ly a sm a l la b s o r p t i o n i n c r e a s e a r o u n d 5 0 0 n m , c h a r a c t e r i s t i c o f o c t a h e d r a l c o m -p lexes . On ly s l i gh t ac t iv a t ion i s obse rved in t h i s p rocess . F u r the r a dd i t i onof two m ore equ iv a l en t s o f Co 2+ r e su l t s i n fu l l ac t iv a t ion and in t he

fo rma t ion o f an in t ense v i s ib l e abso rp t ion . Add i t ion in exces s o f fou re q u i v a l e n t s h a s n o f u r t h e r e f f ec t o n a c t i v i t y o r s p e c t r u m .

Somewha t d ive rgen t r e su l t s , bu t a p rac t i ca l ly i den t i ca l f i na l abso rp -t i o n s p e c t r u m , w e r e o b t a i n e d b yAppleburya n d Coleman 123) ,w h o

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C o b a l t II ) i n M e t a l l o e n z y m e s

f o u n d t h a t t w o e q u i v a l e n t s o f C o 2+ s u f fi c e d t o p r o d u c e a f u l l y d e v e l o p e ds p e c t r u m . T h e i r s p e c t r o p h o t o m e t r i c t i t r a t i o n i s i n a c c o r d a n c e w i t h t w oeq ua l ly s t ron g s i tes or a pa i rw ise b in din g of Co ~+.

W hi l e t he que s t ion o f s to i ch iom e t ry r equ i r e s fu r the r c l a r if i ca tion ,t h e r e i s a g r e e m e n t t h a t t h e i n t e n se s p e c t r a l f o r m r e p re s e n t stwo func t ion-al ly im portan t cobalt ionsa t t he ac t ive s i t e o f t he enzyme . Rega rd le s s o fthe ex i s t ence o f o the r speci fi c s i te s , t he spec t r a l p rope r t ie s o f t heCo II)e n z y m e o f f e r t h e p o s si bi li ti es o f s e l e c ti v e l y s t u d y i n g t h e c a t a l y t i c a l l yes sen t i a l m e ta l -b in d ing s i te s il l t h i s enzym e . Th us , t he spec t rum depe ndso n p H , a n d i t s i n t e n s i t y d ec r e as e s c o n c u r r e n t l y w i t h t h e c a t a l y t i c

I J I I J2 -

A

I -

- 1 - .

I I I ~ I I r

B

3 0 0 -

2 0 0 -

100 -

t

3 0 0

[ r r400 500 600 700

; L , m ~

F i g . 1 6 . C i r c u l a r d i c h r o i s m a n d a b s o r p t i o n s p e c t r a o f C o I I ) a l k a l in e p h o s p h a t a s ea n d i ts c o m p l e x w i t h H P O ~ - . A : C i r c u la r d i c h r o i s m . ]3 : A b s o r p t i o n s p e c t r a . ) ,t h e C o I I )- e n z y m e . . . . . ), t h e c o b a lt e n z y m e - p h o s p h a t e c o m p l e x . F r o mAppleburya n d Coleman 123)

ac t iv i t y a s t he ib I-I i s l owered115) .I n o rg a n i c o r t h o p h o s p h a t e , a p r o d u c to f t h e c a t a l y t i c re a c t io n , a c t s a s a r e v e rs ib l e i n h i b i to r w h e n a d d e d t o t h e

a s s a y s y s t e m a t h i g h ib I-I, a n d i t c a n b e c o v a l e n t l y i n c o r p o r a t e d a t l o w e rv a l u e s o f p H i n a r e v e r s a l of th e d e p h o s p h o r y l a t i o n s t e p115 a).P h o s p h a t eb i n d i n g a t h i g h p H r e s u lt s in a s p e c t r a l c h a n g e o f t h eCo II)e n z y m e115, 123),s t ron g ly sugges t ing a c lo se m e ta l - inh ib i to r l i nkage F ig. 16 ).

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S. Liadskog

A r s e n a t e c o m p e t e s w i t h p h o s p h a t e a n d p r o d u c e s a s pe c if ic s p e c t r a lch nge (124).

W h e r e a s Sim~bsona n d Vallee r e p o r t t h a t two ~ho@hate ionsare re -q u i r e d t o p r o d u c e t h e s p e c t r a l c h a n g e ,A p p l e b u r ya n d Colemanf in d t h a tone i s suff ic ien t(723)a n d t h a t t w o m e t a l i o n s p e r d i m e r a re n e c es s a r y f o ri t s b i n d i n g (724) .T h i s l a t t e r s t o i c h i o m e t r y i s i n a c c o r d a n c e w i t h i n d e -p e n d e n t o b s e r v a t i o n s o n p h o s p h a t e b i n d i n g t o t h e n a t i v e e n z y m e(770)as we l l a s t he k ine t i c r e su l t s i nd i ca t i ng t he p r e sence o f one ac t i ve s i t ep e r d i m e r (772, 773).A s n a t i v e p r e p a r a t i o n s c o n t a i n i n g a b o u t t h r e ee q u i v a l e n t s o f Z n 2+ w e r e f o u n d t o b i n d u p t o 1 .4 H P O 2 - p e r d i m e r(724),s o m e o f t h e o b s e r v e d d i s c r e p a n c i e s m a y b e d u e t o t h e d i f f e r e n t m e t a lc o n t e n t s e m p l o y e d . T h e e x t l a b o u n d p h o s p h a t e m a y b e u n r e l a t e d

t o t h e a c t i v e s it e, b u t f u r t h e r e x p e r i m e n t s a r e n e e d e d to s e t t le th i sp o i n t .

O n t h e o t h e r h a n d , a lk a li n e p h o s p h a t a s e m a y h a v e t w o e q u i v a l e n ta c t i v e s i t e s w h i c h a r e c o u p l e d s o t h a t , n o r m a l l y, o n l y o n e c a n o p e r a t ea t a t i m e . T h i s s e e m s a n a t t r a c t i v e a l t e r n a t i v e f o r a n e n z y m e c o n si s ti n go f t w o i d e n t i c a l s u b u n i t s . I n a p r e l i m i n a r y p a p e r,Lazdunski et al . (725)r e p o r t t h e c o v a l e n t i n c o rp o r a t i o n o f t w o p h o s p h a t e s i n t o t h e z in c e n z y m ea s w e ll a s t h e c o b a l t e n z y m e , a t p H < 4 . A t t h e s e lo w p H v a lu e s, t h e f r e ee n z y m e g e n e r a l l y l o s e s i t s m e t a l i o n s a n d d i s s o c i a t e s i n t o m o n o m e r e s

(709).H o w e v e r, i f t h e s e r e s u lt s a r e c o r r o b o r a t e d a f t e r t h e p e r f o r m a n c e o fp r o p e r c o n t r o ls , a n d i f b o t h p h o s p h a t e s a r e l i n k e d t o s p e ci fi c a m i n o a c i dr e s i d u e s i n t h e e n z y m e , c o n d i t i o n s m a y h a v e b e e n f o u n d f o r t h eu n -c o u p l i n g o f a c t i v e s i te s i n a l k a li n e p h o s p h a t a s e .

T h e circular diehroie sp ectrumof cob a l t a l ka l ine pho sph a t a se (F ig . 16 )s h o w s m o r e c l e a r l y t h e c o m p l e x i t y o f t h e v i s i b le a b s o r p t i o n . A l t h o u g hi t c a n n o t b e r u l e d o u t t h a t t h e s p e c t r u m o f t h isCo II)e n z y m e r e p r e s en t st w o s l i g h t l y d i f f e r e n tco n) s i t e s , t he r e a r e s t r i k ing s imi l a r i t i e s w i thCo II) c a r b o n i c a n h y d r a s e , w h i c h h a s o n l y o n e m e t a l - b i n d i n g s i te . A th i g h / ~ H , c o b a l t c a r b o n i c a n h y d r a s e a n d c o b a l t a l k a l i n e p h o s p h a t a s eh a v e s e v e ra l s p e c t ra l f e at u r e s i n c o m m o n , a n d b o t h m a y h a v e a s i m i l a rk i n d o f i r r e g u l a r c o o r d i n a t i o n . I t s h o u l d b e n o t e d , h o w e v e r, t h a t t h eabs o rp t i on coe ff i ci en t f o rCo n) a l k a l i n e p h o s p h a t a s e p e r e q u i v a l e n t o fa c t i v i t y - l i n k e d m e t a l i o n i s o n l y h a l f o f t h e v a l u e f o rco n) c a r b o n i ca n h y d r a s e .

If E . colii s g r o w n i n t h e p r e s e n c e o f r e l a t i v e l y h i g h c o n c e n t r a t i o n s(3.5 ~M) o f 60Co2+ , an ac t i ve 60Co(I I) enz ym e is sy n th e t i ze d(722).I t sp r o p e r t i e s r e s e m b l e t h o s e o f t h e C o ( II ) e n z y m e r e c o n s t i t u t e d f r o m t h e

a p o e n z y m e . C e r t a i n m u t a n t s o fE. coli p r o d u c e d e f e c t i v e a l k a l i n ep h o s p h a ta s e s . T h e C o ( II ) e n z y m e f r o m t h e m u t a n t U 4 7 l ac k s th e c h a r-a c t e r is t i c b a n d s o f t h e C o ( I I ) - c o m p l e x o f t h e n o r m a l e n z y m e , a n d t h es p e c t r u m is b r o a d a n d p o o r l y r e s o l v e d ( 12 3).

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C o b a l t l l ) n M eta lloenzymes

D . A l c o h o l D e h y d r o g e n a s e

A l c o h o l d e h y d r o g e n as e s , c a t a ly z i n g t h e N A D - d e p e n d e n t o x i d a t i o n o fa l co h o l s t o a l d e h y d e s , a r e z i n c m e t a l l o e n z y m e s 3 ). T h e e n z y m e i s o l a t e df r o m h o r s e l iv e r c o n t a i n s t w o c a t a l y t i c a l l y f u n c t i o n a l z i n c i o n s p e rm o l e c u l e , a n d t w o a d d i t i o n a l z i n c i o n s a r e b e l i e v e d t o h a v e a s t r u c t u r a lro l e 726).I t h a s n o t b e e n p o s s ib l e t o p r e p a r e a s t a b l e a p o e n z y m e , b u texch ang e o f 65Zn~+ fo r t he na t i ve Z n 2+ has bee n ach i eved 127 ). R ecen t ly,pa r t i a l sub s t i t u t i on o f Co s+ fo r Z n 2+ inyeasta l c o h o l d e h y d r o g e n a s e h a sb e e n a c c o m p l i s h e d b y g r o w i n g y e a s t i l l t h e p r e s e n c e o f a n e x c e s s o fadd ed Co s+ 728).T h e e n z y m e i s o l a t e d f r o m t h i s c u l t u r e is g r e en , a n d i ti s a t l e a s t a s a c t i v e a s t h e n a t i v e e n z y m e728).T h i s f i n d in g s h o u l d o p e nn e w p o s s ib i li ti e s o f s t u d y i n g t h e r o l e of t h e m e t a l i o n i n s u b s t r a t e , c o e u -z y m e a n d i n h i b i t o r bi n d in g .

T h e a b s o r p t i o n s p e c t r u m s h o w s th r e e p o o r l y r e s o l v e d p e a k s a t 6 2 0 ,6 7 0 a n d 7 1 0 n m F ig . 1 7). T h e m a x i m a l m o l a r a b s o r b a n c e w a s a b o u t1 0 00 M - 1 c m - 1 f o r t h e p a r t i a l ly s u b s t i t u t e d p r e p a r a t i o n w h i c h m a k e s3 0 0 0 M - 1 c m - 1 p e r e q u i v a l e n t o f c o b a lt . T h e l a rg e i n t e n s i t y a n d t h ep o s i t i o n o f t h e a b s o r p t i o n b a n d s a t l o w e r f r e q u e n c ie s t h a n f o r t h e o t h e rCo II)e n z y m e s d i s cu s s e d a r e s t r ik i n g f e a t u r e s o f t h i s s p e c t r u m , s u g g e s t-i n g a n i r r e g u l a r c o o r d i n a t i o n o fCo II)i n a r e l a t i v e ly wea k f i eld , p r e -s u m a b l y i n v o l v i n g s u l f u r l i g a n d s . A b s o r p t i o n b a n d s i n t h e n e a r u l t r a -v i o l e t a r e m e n t i o n e d 128)w h i c h i n d i c a t e s t h a t c h a rg e - t r a n s f e r b a n d sm a y e x i s t . T h e a v a i l a b l e p r e l i m i n a r y d a t a t o n o t a l l o w a n y d e t a i l e di n t e r p r e t a t io n , a n d f u r t h e r s p e c t ra l a n d m a g n e t i c in f o r m a t i o n w o u l d b ed e s ir a b le . A n o c t a h e d r a l c o o r d i n a t i o n , w h i c h w a s p r o p o s e d f o r t h e

A0.6

0.4

0.2

Co, Z n) -AD H1

Zn-ADHx,~

I I I ~ ~ L J I ~ I T 5 I

6 0 0 7 0 0 8 0 0 n m

Fig . 17. Vi s ib l e abso rp t ion spec t r a o f pa r t i a l l y subs t i t u t ed coba l t yea s t a l coho ldehydrogenase , Co , Zn) -ADH , and na t ive yeas t a lcoho l dehydrogenase, Zn -ADH ) ,respec t ive ly. Enzy m e conc ent ra t ion , a pprox . 0 .35 miVi. Fr omurdel a n d Iwatsubo728)

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S. Lindsko g

c a t a l y t i c a l l y f u n c t i o n a l z in c i on s i n t h e h o r s e li v e r e n z y m e129),a p p e a r sun l ike ly.

E P h o s p h o g l u c o m u t a s e

P h o s p h o g l u c o m n t a s e , c a t a l y z i n g t h e i s o m e r i z a t i o n b e t w e e n g l u c o s e - l -p h o s p h a t e a n d g l u c o se - 6 - p h o sp h a t e v i a a g l u c o s e - l , 6 - d ip h o s p h a t e i n t e r -m e d i a t e , h a s v e r y i n t e r e s t i n g m e t a l - b i n d i n g p r o p e r t i e s . T h e e n z y m e a sn o r m a l l y is o la t e d is p a r t i a ll y i n h i b it e d b y s t ro n g l y b o u n d h e a v y m e t a lion impu r i t ie s . Th e apo enzy m e i s mos t e ff i c i en t ly ac t iv a t ed by Mg 2+,a n d w i t h t h i s w e a k l y b o u n d i o n, t h e e n z y m e b eh a v e s a s a m e t a l - a c t iv a t e dsys tem . A ct iv i ty i s a l so ob ta in ed w i th Ni 2+, Co 2+, M n 2+, Cd 2+ or Zn =+130).M o s t o f t h e s e io n s b i n d q u i t e s t ro n g l y, s o t h a t t h e e n z y m e b e c o m e s

a m e t a l l o e n z y m e a c c o r d i n g t o t h e u s u a l o p e r a t i o n a l d e f i n i t i o n . I n ase ri es o f pape r s , desc r ib ing the e ff ec ts o f va r ious m e ta l i ons on the ac t iv i t ya n d u l t r a v i o l e t a b s o r p t i o n s p e c t r u m o f t h e e n z y m e ,R a y 1 3 0 )a n d R a ya n d Peck 137 , 132)h a v e a ls o s t u d i e d C o I I ) - p h o s p h o g l u c o m u t a s e . I t ism e n t i o n e d t h a t t h e c o b a l t e n z y m e h a s a m e a s u r a b l e v i si b le a b s o r p t i o n132).T h e s p e c t r u m h a s f e a t u r e s r e m i n i s c e n t ofCo(II)a l k a l in e p h o s p h a -

t a s e o r c o ( n ) c a r b o n ic a n h y d r a s e , a n d i t is s e n s it iv e to p H a n d t h e a d d i -t i o n o f s u b s t r a t e s a n d p s e u d o s u b s t r a t e s133).

F O ther Cobalt Prote ins

A n u m b e r o f o t h e r c o b a l t e n z y m e s h a v e b e e n p r e p a r e d . T h e i r p h y s ic a lp r o p e r ti e s h a v e n o t y e t b e e n s t u d i e d , h o w e v e r, a n d t h e i n f o r m a t i o n isr e s t r ic t e d t o t h e i r c a t a l y t i c b e h a v i o r.

T h e f l a v o e n z y m e D - l a c ta t e d e h y d r o g e n a s e f ro m y e a s t h a s b e e n r e p o r t -

e d t o c o n t a i n z in c 1 34 ). A n a p o e n z y m e c a n b e p r e p a r e d a n d r e a c t i v a t e dby Zn 2+ o r CoZ+135) .W h e n y e a s t i s g r o w n i n t h e p r e s e n c e o f a d d e dC o a Co(II)e n z y m e i s s y n t h e s i z e d . T h e b i o s y n t h e t i cCo(II) e n z y m ew a s f o u n d to h a v e d i f f e re n t c a t a l y t i c p ro p e r t ie s c o m p a r e d t o t h e e n z y m er e a c t i v a t e d f r o m t h e a p o e n z y m e736) .O n l y r a t h e r f r a g m e n t a r y d a t ahav e been pub l i shed on th i s sub jec t , an d the d i f fe r ences i n coba l t b ind ingo b t a i n e d b y t h e t w o m e t h o d s o f p r e p a r a t io n a r e u n k n o w n .

A n a c t iv e , c o b a l t -c o n t a i n in g , o x a l o a c e t a t e t r a n s c a r b o x y l a s e m e t h y l -m a l o n y l - C o A : p y r u v a t e c a r b o x y l t ra n s f e r a s e ) h a s b e e n i s o la t e d f r o m

Propionobacter ium shermaniigrow n w i th 60Co~+137).T h e m e t a l c o n t e n tc o r r e sp o n d s t o t w o e q u i v a l e n t s o f6OCo(II)p e r m o l e o f e n z y m e .

Yeas t a ldo la se i s a z inc me ta l loenzyme738).T h e m e t a l i o n is e a s i lyd i s so c i a te d a n d a c t i v i t y c a n b e r e s t o r e d t o t h e a p o e n z y m e b y Z n 2+,

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Cobalt(II) in Metalloenzymes

Co2+, Ni~+, Mn~.+ and Fe 2+, whereas Cu 2+, Hg 2+, Cd 2+, Mg ~+ and Fe 8+fail to reactivate (7$8).

The zinc ion in a neutral protease from Bacil lus subt i l ishas beenexchanged with other metal ions (739--15]). The co n) enzyme isreported to be active (140).

A cobalt complex of transferrin has been prepared by addition ofco n) citrate to tile apoprotein. Hydrogen peroxide was added to obtainthe absorption spectrum of cobalt transferrin, and susceptibility measure-ments showed that the metal ion was incorporated as diamagnetic Co(In)(142).

IV. Conclu ding Remarks

The most important aspect of the study of Co II)metalloenzymes isthe possibility of using the metal ion as a functional, built-in reporterof the dynamics of the active site. The spectral and magnetic propertiesof co n) carbonic anhydrase have given valuable clues to the catalyticfunction of this enzyme. The recent studies of Co(II) alkaline phosphataseand co n) carboxypeptidase A indicate the general applicability of thisapproach to enzymes where the probe properties of the constitutive

metal ion are poor. The comparison of the absorption spectra of theseenzymes and low-molecular weight models have shown th at the proteinsprovide irregular, and in some cases nearly tetrahedral environments.It is obvious, however, that a knowledge of the crystal structures of theenzymes is necessary before the full potential of this method Call beexploited.

Metal binding in the cobalt enzymes appears to have certain commonfeatures. The te rti ary structure of the proteins provides a chelating sitewhere the metal ion is firmly, but not altogether rigidly, fixed. These

ligands are not easily exchangeable, and the metal ion is par tly bur iedin the protein matrix. At least one coordination position is open towardsthe catalytic center and call be occupied by a solvent molecule in theresting enzyme or by other compounds which are inhibitory because theyinterfere with the function of the metal ion. This function involves thebinding and polarization of participants in the catalytic reaction, andrequires a fast exchange of ligands at the open site.

Possible consequences of the irregular mode of metal binding for thereact ivity of the complex have been fully discussed by Valleeand Wi l l i a m s

(17). It is perhaps worth emphasizing, however, that the cooperationwith other reactive groups not directly associated with the metal ionseems to be of decisive importance for the catalytic action of the metallo-enzymes discussed here.

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S . L indskog

T h e C o ( I I ) e n z y m e s a r e s t il l u n i q u e i n t h e s e n s e t h a t t h e r e a r e a sy e t n o m o d e l s o f k n o w n s t r u c t u r e m i m i c k i n g t h e i r s p e c t ra l p r o p e r t i e ss o a s t o m a k e p o s s i b l e a d i r e c t t r a n s l a t i o n o f c o o r d i n a t i o n n u m b e r ,g e o m e t r y a n d l i g a n d g r o u p s. T h e s p e c t r a l p r o p e r t i e s ofCo II)e n z y m e ss u g g e s t t h a t t h e m o s t u s e f u l m o d e l s f r o m a b i o c h e m i s t ' s p o i n t o f v i e wa r e n o t n e c e s s a r i ly c o m p o u n d s w i t h l i g an d s o f t h e s a m e c h e m i c a l n a t u r ea s e n c o u n t e re d i n t h e p r o t e in s , b u t c o m p o u n d s o f u n u s u a l s t r u c t u r e sa n d g e o m e t r i es . I n f a c t , th e r e s e e m s t o b e a t r e n d i n c o o r d i n a t i o n c h e m -i s t r y t o s e e k fo r n e w a n d s t r a n g e l i g a n d s f o r m i n g c o m p l e x e s o f d i f f e re n ts t e r e o c h e m i s t r i e s . I t i s h o p e d t h a t t h i s r e v i e w w i ll h e l p t o i n t e r e s tc o o r d i n a t i o n c h e m i s t s i n t h e b i o c h e m i c a l a s p e c t s of t r a n s i t i o n m e t a lc h e m i s t r y a n d t o s t i m u l a t e t h e m t o a p p l y th e i r k n o w l e d g e of l i g a n d

f ie ld t h e o r y a n d s p e c t r o s c o p y t o e v e r m o r e c o m p l i c a t e d c o m p l e xe s , e v e n -t u a l l y a l so t h e m e t a l l o e n z y m e s .

V R e f e r e n c e s

1. Hogenkamp H. P . C. : Ann. Rev. Biochem. 37, 225 (1968).2. Hill J. A. Pratt J. M. Williams R. ]. P.: J. Chem. Soc. 5149 (1964).

3. Vallee B. L. Coleman f . E . : In : Comprehens ive B iochemis t ry12 164. Ed .b y M. Florkin a n d E. H. Stotz. A m s t e r d a m - L o n d o n - N e w Yo r k : E l s ev i e r 19 64 .

4. Smi th .E. L. : In : Mine ra l M e tabo l i sm2 Part B 349. Ed . byC. L. Comar a n dF. Bronner. New Y ork -L ond on : Academ ic P re ss , I nc . 1962 .

5. Malmstr6m B. G. Rosenberg A : Advan . Enzymol .27 131 (1959).6. Williams R. J. P . : E n z y m e s 7 391 (1959).7. Vallee B. L. Riordan f . F. : Ann. Rev. Biochem.38 733 (1969).8. Cohn M . : In : Magne t i c Resonance in B io log ica l Sys t ems , p . 101 . Ed . by

A. Ehrenberg B. G. MalmstrSm a n d T. VSnngdrd. Oxford : Pe rgam on P re s s,Inc. 1967.

9. Vallee B. L. Rupley J. A. Coombs T. L. Neurath H. : J. Am. Chem. Soc.80

4750 (1958).10. Orgel L. . : An In t rod uc t ion to Trans i t i on -Meta l Chem is t ry : L igand -F ie ldTheory, p . 71 . Lon don : M ethuen Co., L td . 1960.

11. Freeman H. C. : Adv an . P ro t e in Chem.22 258 (1967).12. Cotton F. A. Goodgame D. M. L. Goodgame M . : J . Am . Chem. Soc.83 4690.

(1961).13. Ciampolini M. : Struct . Bonding 6, 52 (1969).14. Hare C. R. : In : Spec t roscopy and S t ruc tu re o f Me ta l Che lat e Compounds ,

p . 73 . Ed . byK. Wakamoto and P. J. McCarthy. N e w Yo r k : J o h n Wi l e ySons, Ltd. 1968.

15. Carlin R. L . : In : Trans i t i on Me ta l Chemis t ry 1 , 1. Ed . byR. L. Carlin. N e w

York: Marce l Dekker 1965.16. Dennard A. E. Williams R. J. P. : In : Trans i t i on Me ta l Chemis t ry2 116.:Ed. by R. L. Carlin. New York: Marce l Dekker 1966.

17. Vallee B. L. Williams R. J. P.: Proc . Nat l . Acad . Sc i . U.S .59 498 (1968).18. Ballhausen C. J. Jorgensen c. K. : A cta Chem. Scand.9 397 (1955).

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19. C o t to n F . A . G o o d g a m e D . M . L . G o o d g a m e M . S a co oA . : J . A m . C h e m .Soc. 83 4157 1961) .

20 . Goodgame D. M . L . Goodgame M . :I n o rg . C h e m . 4 , 1 3 9 1 9 6 5 ).21 . C i a m p o l i n i M . N a r d i N . :I n o r g . C h e m . 6 445 1967) .22 . B a y e r E . S e h r e t z m a n n P. :S t r u c t . B o n d i n g 2, 1 81 1 9 6 7 ).23 . H u n t e r S . H . R o d l e y G . A . :I n : P r o c e e d i n g s o f t h e X I I . I n t e r n a t i o n a l C o n -

f e re n c e o n C o o r d i n a t i o n C h e m i s tr y, p . 4 . E d . b yH . C . F r e e m a n . S y d n e y :S c i e n c e P r e s s 1 9 6 9 .

24. C o c kl e S . A . H i l l H . . 4 . 0 . P r a t t J . M . W i l l i a m s R . J . P. :B i o c h i m . B i o p h y s .A c t a 177 686 1969) .

25 . H a m i l to n J . A . B l a k l e y R . L . L o o n e y F. D . Wi n f i el d M . E . :B i o c h i m .B i o p h y s . A c t a177 374 1969) .

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