METAL COORDINATION IN ZINC ENZYMES

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METAL COORDINATION IN ZINC ENZYMES

M. Feiters, C. Little, S. Waley

To cite this version:

M. Feiters, C. Little, S. Waley. METAL COORDINATION IN ZINC ENZYMES. Journal de Physique

Colloques, 1986, 47 (C8), pp.C8-1169-C8-1172. �10.1051/jphyscol:19868228�. �jpa-00226212�

J O U R N A L D E P H Y S I Q U E

C o l l o g u e C8, s u p p l e m e n t a u n o 1 2 , Tome 47, d e c e m b r e 1986

METAL COORDINATION IN ZINC ENZYMES

M.C. F E I T E R S * * " * , C . LITTLE***(^) a n d S . G . W A L E Y " " '

'~epartment of Chemistry, University of Manchester, GB-Manchester M13 9 P L , Great-Britain

""Synchrotron Radiation Source, SERC Daresbury Laboratory, GB-Warrington WA4 4 A D , Great-Britain

" * s i r William Dunn School of Pathology, University of Oxford, South Parks Road, GB-Oxford OX1 3 R E , Great-Britain

R & S U ~ ~

Nous avons dtudi6 des exo-enzymes de B a c i l l u s Cereus, phospho1ipase-C e t

@-lactamase 11, par zinc e t c o b a l t K-edge EXAFS. Nous rapportons que l e s zincs dans l e s c e n t r e s t r u c t u r a l e t c a t a l y t i q u e de phospholipase-C o n t cinq l i g a n t s , t a n d i s que l e c o b a l t , s u b s t i t u 6 dans l e c e n t r e c a t a l y t i q u e , a s i x l i g a n t s . Le zinc dans l e c e n t r e a c t i f de $-lactamase 11 a un l i g a n t soufre, $ bas nombre de coordination ou haut f a c t e w Debye-Waller, o u t r e des l i g a n t s imidazoles.

Abstract

W e have s t u d i e d t h e B a c i l l u s Cereus exo-enzymes, phospholipase-C and p-lactamase 11, by Zn and Co K-edge EXAFS. W e r e p o r t t h a t the zincs i n the s t r u c t u r a l and c a t a l y t i c s i t e s of t h e former enzyme a r e 5-coordinate, whereas c o b a l t , s u b s t i t u t e d

1

i n t h e c a t a l y t i c s i t e , i s 6 coordinate. The p-lactamase 11 a c t i v e s i t e zinc has sulphur coordination, with e i t h e r low occupancy or high Debye-Waller f a c t o r , i n a d d i t i o n t o imidazole coordination.

A s zinc i s normally spectroscopically i n a c c e s s i b l e , zinc enzymes have o f t e n been s t u d i e d by s u b s t i t u t i o n of t h e zinc by c o b a l t , a process which usually r e t a i n s enzyme a c t i v i t y t o some e x t e n t 1 1 1 . Apart from crystallography, which i s not always applicable, EXAFS i s t h e only technique by which zinc and cobalt-substituted enzymes can be compared and t h e v a l i d i t y of e x t r a p o l a t i o n of t h e spectroscopic s t u d i e s of c o b a l t enzymes t o zinc enzymes checked. For example, c r y s t a l l o g r a p h i c s t u d i e s suggest 4-coordination f o r zinc i n carbonic anhydrase, whereas W/vis spectroscopic s t u d i e s suggest t h a t c o b a l t i n t h i s enzyme is 4- or 5-coordinated, depending on pH and presence of i n h i b i t o r s . X-ray absorption s t u d i e s give d e f i n i t e evidence t h a t d i f f e r e n c e s between zinc and c o b a l t - s u b s t i t u t e d carbonic anhydrase e x i s t I21.

("on leave from Institute of Medical Biology. University of Tromsa, N-9000 TromsZ. Norway

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19868228

C8-1170 JOURNAL DE PHYSIQUE

For B a c i l l u s c e r e u s phospholipase C, chemical m o d i f i c a t i o n s t u d i e s have i n d i c a t e d t h e absence of s u l p h u r l i g a t i o n , and involvement of f o u r h i s t i d i n e l i g a n d s , p o s s i b l y one b r i d g i n g , f o r t h e two z i n c atoms [ 3 1 , which a r e 5.7 8 a p a r t [4]. S e l e c t i v e s u b s t i t u t i o n by c o b a l t is f e a s i b l e , and s p e c t r o s c o p i c s t u d i e s s u g g e s t t h a t t h e c o b a l t - s u b s t i t u t e d s i t e s a r e s i m i l a r , i n t e r a c t w i t h each o t h e r , and have d i s t o r t e d o c t a h e d r a l l i g a n d geometry [ 5 ] . We have i n t e r p r e t e d t h e Zn-EXAFS of t h e s t r u c t u r a l and c a t a l y t i c s i t e s , and t h e Co-EXAFS of t h e l a t t e r , and compared t h e r e s u l t s . These s u g g e s t t h a t z i n c i s 5-coordinated i n both sites, w i t h 3 N(His) l i g a n d s a t 2.01-2.02 8 and 2 N o r 0, p o s s i b l y water oxygens, a t 2.13-2.15 a (Fig. 11, whereas c o b a l t , i n agreement with t h e s p e c t r o s c o p i c s t u d i e s , has 6-coordination i n t h e c a t a l y t i c s i t e ( F i g . 2).

Fig. 1. EXAFS (upper p a n e l ) and Fig. 2. As Fig. 1, b u t t h e s o l i d and p h a s e s h i f t - c o r r e c t e d modulus of t h e dashed l i n e s r e p r e s e n t Co and Zn, F o u r i e r t r a n s f o r m (lower p a n e l ) r e s p e c t i v e l y , i n t h e phospholipase-C experimental ( s o l i d l i n e ) and c a t a l y t i c s i t e .

s i m u l a t i o n (dashed l i n e ) of Zn in

phospholipase-C, s t r u c t u r a l s i t e .

For t h e B.cereus p-lactamase 11 a c t i v e s i t e , t h e r e i s evidence f o r t h r e e h i s t i d i n e l i g a n d s and a s u l p h u r l i g a n d [61. However, t h e i n t e n s i t y of t h e Co-S c h a r g e t r a n s f e r band is r a t h e r low compared t o t h a t of o t h e r c o b a l t - s u b s t i t u t e d p r o t e i n s with s u l p h u r l i g a t i o n I71. T h i s seems t o b e r e f l e c t e d i n t h e Zn-EXAFS, which can be simulated with h i s t i d i n e l i g a n d s alone. A sulphur l i g a n d , when included i n t h e s i m u l a t i o n , comes a t a normal Zn-S d i s t a n c e , b u t with e i t h e r a h i g h Debye-Waller f a c t o r o r a low occupancy ( F i g . 3 ) .

Fig. 3. As Fig. 1, b u t f o r Zn i n the p-lactamase I1 a c t i v e s i t e , t h e dashed l i n e

r e p r e s e n t i n g s i m u l a t i o n 1 of Table 1.

JOURNAL DE PHYSIQUE

Table 1. Parameters u s e d f o r t h e s i m u l a t i o n s

D i s t a n c e s in 8, Debye-Waller f a c t o r s i n b r a c k e t s a s a = 2a2 i n A2, F i , f i t index;

N , n i t r o g e n ; 0, oxygen; S, s u l p h u r ; C, carbon.

Phosphol ipase-C @-lactamase 11

S t r u c t u r a l C a t a l y t i c Simulation 1 Simulation 2 N 3 a t 2.01 3 a t 2 . 0 1 4 a t 2.02 5 a t 2.02

(0.004) (0.003) (0.010) (0.015)

Acknowledgement

The a u t h o r s t h a n k t h e D i r e c t o r of t h e SEX Daresbury Laboratory f o r p r o v i d i n g experimental f a c i l i t i e s f o r t h e EXAFS s e r v i c e .

References

[ I ] References (1983) in Metal I o n s in Biology (T.G. S p i r o , e d i t o r ) Vol.IV: Zinc Enzymes (Wiley: New York).

[ 2 ] Yachandra, V., Powers, L. and S p i r o , T.G. (1983 ) J. Am. Chem. Soc. 105,

6596-6604.

[3] L i t t l e , C. (1977) Biochem. J. 167, 399-404.

[4] Aalmo, K.H., Hansen, L., Hough, E., Jynge, K., m a n e , J., L i t t l e , C. and Storm, C.B. (1984) Biochem. I n t . 2, 27-33.

[5] B i c k n e l l , R., Hanson, G.R., Holmquist, B. and L i t t l e , C. (1986) Biochemistry, submitted f o r p u b l i c a t i o n .

[61 H i l l , H.A.O., Sammes, P.G. and Waley, S.G. (1980) P h i l . Trans. R. Soc. Lond.

B X , 333-344.

[71 Baldwin, G.S., Galdes, A., H i l l , H.A.O., Waley, S.G. and Abraham, E.P. (1980) J.

Inorg. Biochem. 13, 189-204.