EXAFS INVESTIGATION OF THE STRUCTURAL SITE OF LIVER ALCOHOL DEHYDROGENASE

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EXAFS INVESTIGATION OF THE STRUCTURAL SITE OF LIVER ALCOHOL DEHYDROGENASE

M. Zeppezauer, C. Haas, W. Maret, C. Hermes, R. Pettifer

To cite this version:

M. Zeppezauer, C. Haas, W. Maret, C. Hermes, R. Pettifer. EXAFS INVESTIGATION OF THE

STRUCTURAL SITE OF LIVER ALCOHOL DEHYDROGENASE. Journal de Physique Colloques,

1986, 47 (C8), pp.C8-1165-C8-1168. �10.1051/jphyscol:19868227�. �jpa-00226211�

Colloque C8, supplement au n o 1 2 , Tome 47, decembre 1 9 8 6

EXAFS INVESTIGATION OF THE STRUCTURAL SITE OF LIVER ALCOHOL DEHYDROGENASE

M. ZEPPEZAUER, C. HAAS, W. MARET, C. HERMES* and R.F. PETTIFER*

Fachbereich 15.2, Analytische und Biologische Chemie, Universitst des Saarlandes, 0-6600 Saarbrucken, F.R.G.

" ~ u r o p e a n Molecular Biology Laboratory (EMBL), C/o DESY, Notkestrasse 85, 0-2000 Hamburg 52, F.R.G.

A b s t r a c t

Data a r e presentee of t h e X-ray a b s o r p t i o n n e a r edge a n d CXAFS r e g i o n f o r t h e Zinc environment of t h e s t r u c t u r a l s i t e of h o r s e l i v e r a l c o h o l dehydrogenase (IILADI!) . Using m e t a l r e p l a c e n e n t o r e x t r a c t i o n , X-ray a b s o r p t i o n n e a s u r e n e n t s c o u l d be performed e x c l u s i v e l y on t h e s t r u c t u r a l s i t e . r i e a s u r e n e n t s were performed a t 20 ! ; b o t h on 3 t y p e s of enzyme (iTLADH ~ - z i t l ~ o u t c a t s l y r i c c e n t r e s , 13LADC w i t h coenzyme and I'LADII i l i t h i n h i b i t o r bound t o t h e a c t i v e s i t e ) and on 2 rtlodel compounds n a n e l y c u b i c Zinc s u l p h i d e and Zinc dinethyldithiocarbanate.

Both, t h e mean i n t e r a t o m i c d i s t a n c e (Zn-S) and t h e t h e r m a l p a r a m e t e r s i n c l i c a t e t h a t t h e e n z y n e s ' l o c a l s t r u c t u r e i s n o s t c l o s e l y modelled by c u b i c Zinc s u l p h i d e .

A s p a r t of an e x t e n s i v e s t u d y of t h e enzyme h o r s e l i v e r a l c o h o l dehydrogenase (1:LADI:) we p r e s e n t h e r e th'e r e s u l t s of a s t u d y of t h e s t r u c t u r a l s i t e i n t h i s enzyne. The enzyne i s a d i m e r i c m o l e c u l e v i t h two t i g h t l y bound Zn-atoms p e r s u b u n i t ( m o l e c u l a r w e i g h t p c r s u b u n i t 4 0 0 0 0 D a l t o n s ) . The m e t a l - s i t e s t r u c t u r e s oE t h e s e i o n s a r e known from X-ray c r y s t a l l o q r a p h y and i n c l u d e a s t r u c t u r a l Zinc t h o t i s c o o r d i n a t e d t o f o u r c y s t e i n a t e s and a c a t a l y t i c Zinc t h a t i s c o o r d i n a t e d t o two c y s t e i n a t e s , one h i s t i d i n e , and one water molecule

[ I ] . I t h a s been found t h a t t h e z i n c i n t h e c a t a l y t i c s i t e c a n be r e p l a c e d by c o b a l t w h i l s t r e t a i n i r l g i t s c a t a l y t i c a c t i v i t y 121. Thus, i t i s p o s s i b l e t o s t u d y both s i t e s of t h e enzycle. The c a t a l y t i c s i t e monitored a t t h e Co K-ecige and t h e s t r u c t u r a l s i t e a t t h e Zn I<-edge.

I t i s p o s s i b l e t o a t t a c h l i g a n d s t o t h e a c t i v e s i t e , e.g. t h e coenzyrne IIADI: o r t h e i n h i D i t o r o r t l i o p h e n a n t h r o l i n o r t o remove t h e a c t i v e s i t e c o m p l e t e l y 131. T.e have ~neasurecl t h e Zinc X-ray a b s o r p t i o n s p e c t r a of t h e s e t h r e e t y p e s of enzynes t o g e t h e r w i t h two

model s y s t e r i ~ s namely c u b i c Zinc s u l p h i d e and Zinc

d i m e t h y l u i t h i o c a r b a r ~ a t e .

A 1 1 n e a s u r e n e n t s were p e r f o r ~ l e d a t 2 0 I< on t h e CXAFS a p p a r a t u r s [ 4 l of EliRL s i t u a t e d i n tiASYLAB on t h e s t o r a g e r i n g D O R I S 11. The s ? e c t r a were c a l i b r a t e d a b s o l u t e l y [ 5 1 , which a l l o w e d d a t a t o be conpareci

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19868227

CX-1166 JOURNAL DE PHYSIQUE

which were t a k e n o v e r a p e r i o d of two y e a r s . The enzyne m a t e r i a l as f r e e z e d r i e d a n d packed i n t o c e l l s r r i t h t h i n !Captoxi rlincior~s.

F l u o r e s c e n c e s i g n a l s w e r e d e t e c t e d u s i n g two f a s t p l a s t i c s c i n t i l l a t o r s mounted a t 90° t o t h e i n c i d e n t bean, which was a S j j u s t e d f o r naximum o - p o l a r i s a t i o n . ( F u r t h e r d e t a i l s w i l l be p u b l i s h e d e l s e w h e r e ) . The sar.iples w e r e exposed f o r one h o u r p e r s p e c t r u m , a n b f o r a v e r a g i n g a f r e s h s e c t i o n o f s a m p l e was e x p o s e d u s i n 5 a r e m o t e l y c o n t r o l l e d t r a n s l a t i o n d e v i c e i n s i d e t h e c r y o s t a t . T h i s p r o c e d u r e , t o g e t h e r w i t h t h e c r y o g e n i c t e m p e r a t u r e s h e l p s t o n i n i m i s e r a d i a t i o n d a a a g e [61. A f t e r f u l l e x p o s u r e of t h e enzyme s p e c i m e n s , t h e c a t a l y t i c a c t i v i t y was c h e c k e d a n d f o u n d t o be o n l y r e d u c e d by 1 0 % . The two model s y s t e m s w e r e measured i n t r a n s m i s s i o n a t 2 0 I<.

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F i g . 1 shows t h e n o r r ~ a l i s e d Z i n c I<-edge s p e c t r a of t h e t h r e e e n z y n e s p e c i m e n s t o g e t h e r w i t h t h e two s t a n d a r d m a t e r i a l s . The h a l f o s c i l l a t o r - s t r e n g t h p o i n t s o f a l l e d g e s a r e c o i n c i d e n t t o w i t h i n

f. 0.1 eV, which c o r r e s p o n u s t o t h e a c c u r a c y of o u r e n e r q y c a l i b r a t i o n . T h i s c o n f i r m s t h a t l i g a n t i s o t h e r t h a n s u l p h u r a r e n o t bonded t o t h e s t r u c t u r a l s i t e . On t h e o t h e r hand, t h e XANCS s t r u c t u r e i s c o n s i s t e n t between t h e enzyrnes b u t d i f f e r e s from t h e two s t a n d a r d s a n d shows t h a t t h e XAIJES i s v e r y s e n s i t i v e t o l i g a n d s beyond t h e f i r s t s h e l l . For t h e enzyme s p e c i m e n s t h i s c l e a r l y d e m o n s t r a t e s t h a t no m a j o r s t r u c t u r a l c h a n g e s o c c u r o u t s i u c t h e f i r s t c o o r d i n a t i o n s p h e r e upon

F i g . 1

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A l l s p e c t r a were r e c o r d e d a t 2 0 K .

coenzyme b i n d i n g t o t h e ? . c t i v e s i t e , rer.lova1 of t h e m e t a l c e n t r e s , o r upon b i n d i n g a n i n h i b i t o r t o t h e a c t i v e s i t e .

The EXAFS f i n e s t r u c t u r e s a r e shown i n P i g . 2 and t h e F o u r i e r

t r a n s f o r m s of t h i s d a t a i n F i g . 3 . i Q a r t from t h e c u b i c Z i n c

su1phi.de specimen, no s c a t t e r i n g c a n be o b s e r v e d beyond t h e f i r s t

s h e l l . However, a c l o s e r i n s p e c t i o n of F i g . 2 shocrs t h a t

d i m e t h y l d i t h i o c a r b a n ~ a t e h a s a l o v e r a m p l i t u d e s i g n a l and ( a t h i g h k ) a

s l i g h t s h i f t of p h a s e w i t h r e s p e c t t o t h e enzyme d a t a . A more

d e t a i l e d i n v e s t i g a t i o n oT t h i s f i n d i n g i s p o s s i b l e by u s i n g t h e now

s t a n d a r d t e c h n i q u e of b a c k t r a n s f o r r n i n g t h e f i r s t s h e l l c o n t r i b u t i o n s

a n 2 comparing t h e a m p l i t u d e s and p h a s e s [71.

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i n F i g . 1. shown i n F i g . 2 .

P h a s e a n d a n p l i t u d e p l o t s a r e shown i n F i g . 4 and F i g . 5 w i t h c u b i c Z i n c s u l p h i d e a s r e f e r e n c e m a t e r i a l . I t i s c l e a r f r o m P i g . 4 t h a t f o r a l l enzyme s p e c i m e n s t h e s u l p h u r l i c j a n d s a r e l o c a t e d a t a s l ' g h t l y l a r g e r r a d i a l d i s t a n c e t h a n . i n c u b i c Z i n c s u l p h i d e (2.340 1). ^A

d i E i e r e n c e o f ( 3 + ^{2 ) x l o m 3 A} i s f o u n d , i n d e p e n d e n t o f u h a t l i g z n i l s a r e bound t o t h e a c t i v e s i t e . On t h e o t h e r h a n d , t h e d i m e t h y l d i t h i o c a r b a 9 a t e s p e c t r u m g i v e s a f i r s t s h e l l r a d i u s w h i c h i s ( 2 6 +_ 2 ) x A l a r g e r . P i g . 5 i l l u s t r a t e s a f u r t h e r s i m i l a r i t y o f t h e c u b i c Z i n c s u l p h i d e a n d enzyme d a t a . I t s h o v s t h a t t h e mean p u a r e r e l a t i v e d i s p l a c e m e n t

i n t h e enzymes i s o n l y 1 . 0 x

l a r g e r t h a n i n c u b i c Z i n c s u l p h i d e . I n c o n t r a s t , t h e d i r y t ? l d i t h i o c a r b a r n a t e s p e c t r u m y i e l d s a a 2 - v a l u e , which i s 4.0 x 10- SY l a r g e r .

I t i s p a r t i c u l a r l y s a t s i f a c t o r y t h a t t h e enzyme c u r v e s i n F i g . 5 i n t e r c e p t t h e v e r t i c a l a x i s a l w a y s a t p o i n t s t h a t c o r r e s p o n d t o w i t h i n +. 5 O t o . 4 a t o m s i n t h e f i r s t c o o r d i n a t i o n s h e l l . U n f o r t u n a t e l y , t h e d l n e t h y l d i t h i o c a r b a m a t e d a t a i n d i c a t e a c o o r d i n a t i o n number o f

4.8 ( + 5 % ) . The o r i g i n o f t h i s e r r o r i s unknown, b u t c e r t a i n l y n o t r e l a t e d t o s p e c t r a l i m p u r i t y o f t h e X-ray beam o r s a m p l e i n h o n o g e n e i t y .

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r e f e r s t o t h e numbers g i v e n i n F i g . 4 and As t o c u b i c Z i n c s u 1 p h i . d e . E r r o r b a r s a r e o n l y d r a w n f o r one e n z y m e d a t a s e t . T h e s l o p e and i n t e r c e p t w i t h . t h e Y - a x i s of t h e s e c u r v e s i s r e l a t e d t o t h e d i f f e r e n c e i n r e l a t i v e m e a n s q u a r e d i s p l a c e m e n t a n d t o t h e n u m b e r of f i r s t s h e l l a t o m s r e s p e c t i v e l y .

lle have f o u n d t h a t t h e f i r s t s h e l l e n v i r o n m e n t of t h e s t r u c t u r a l sJte of HLADI-I i s i n v a r i a n t t o l i g a n u s bound t o t h e a c t i v e s i t e or t o t h e a c t i v e s i t e b e i n g r e m o v e d c o n p l e t e l y , and t h a t it i s a n a l o g o u s t o t h e s o l i d s t a t e m o d e l c o r n p o u n d c u b i c Z i n c s u l p h i d e . O n t h e o t h e r hand, Z i n c d i m e t h y l d i t h i o c a r b n n a t e i s a m o l e c u l a r m a t e r i a l a n d c l e a r l y n o t s u c h a close a n a l o g u e a s t h e s o l i d s t a t e ( Z n - S ) m o d e l . T?e b e l i e v e t h a t t h i s r e s u l t s f r o m t h e f a c t t h a t t h e s t r u c t u r a l s i t e i s b o u n d i n t h e i n t e r i o r of t h e e n z y m e an2 c o n s e q u e n t l y t h e e n v i r o n m e n t i s m o r e a k i n t o t h e s o l i d . C o h e s i o n o f t h e m o l e c u l e i s t h u s n o t s o l e l y d o m i n a t e d by t h e Z n - c y s t e i n e bonds b u t a c o n t r i b u t i n g e l e m e n t .

O t h e r w o r k h a s been p e r f o r m e d o n a s i m i l a r s t r u c t u r a l s i t e i n t r a n s c a r b a m y l a s e [ % I . T h e s e a u t h o r s s h o w t h a t t h e r e i s a c l o s e s i m i l a r i t y b e t w e e n t h e e n z y m e e n v i r o n m e n t a n d Z i n c d i n e t h y l d i t i o c a r b a m a t e , h o w e v e r , t h e i r a n a l y s i s was i n s u f f i c i e n t t o s h o w t h e s m a l l d i f f e r e n c e s p r e s e n t e d i n this paper.

O n e of u s (R.F.P.) t h a n k s t h e U n i v e r s i t y of W a r w i c k f o r l e a v e of absence.

T h i s w o r k was s u p p o r t e d by t h e GMFT and t h e DFG.

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