Fluorinated Carbohydrates as Probes of Enzyme Specificity and

Chapter 5

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 12, 2017 | http://pubs.acs.org Publication Date: August 2, 1988 | doi: 10.1021/bk-1988-0374.ch005

Fluorinated Carbohydrates as Probes of Enzyme Specificity and Mechanism Stephen G. Withers, Ian P. Street, and Michael D. Percival Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Y6, Canada The hydrogen bond strengths and polarities at each of the sugar hydroxyls in the glycogen phosphorylase/ glucose complex have been determined through measurement of the affinities of a full series of deoxy and deoxyfluoro analogues of glucose. Comparison of this data with the recently refined X-ray crystal structure of this complex reveals that the strongest hydrogen bonds measured (~3 kcal mol-1) involve charged hydrogen bond partners and that hydrogen bonds between neutral partners contribute some 0.5 to 1.5 kcal mol-1. The electronegative fluorine substituent is also shown to decrease rates of glycoside hydrolysis, presumably by inductive destabilisation of oxocarbonium ion-like transition states. Studies with fluorinated substrates and glycogen phosphorylase have provided evidence for an oxocarbonium ion mechanism and allowed estimation of the strengths of hydrogen bonds involved in stabilising the transition state. A novel mechanism-based inhibitor of β-glucosidases, 2,4-dinitrophenyl-2-deoxy-2fluoro-β-D-glucopyranoside, is also described. This inhibitor works by rapidly glycosylating the enzyme but only very slowly hydrolysing from the active site. Deoxyfluoro- and deoxy-sugars are useful in studies both of binding interactions between carbohydrates and proteins, and in mechanisms of glycosyl transfer. This paper describes how we have used such analogues to measure hydrogen bond strengths in a carbohydrate/ protein complex of known structure. It proceeds to review how such substitutions affect the rates of glycosyl transfer in a non-enzymatic model reaction and then applies this information to the results obtained in our enzymatic system. There are a number of reasons why these analogues are useful. Firstly the substitution of a sugar hydroxyl by a fluorine or by hydrogen is sterically conservative since both these substituents are smaller than the original hydroxyl. Table I provides the relevant 0097-6156/88/0374-0059$06.00/0 • 1988 American Chemical Society

TAYLOR; Fluorinated Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

d a t a and i t i s c l e a r here t h a t w h i l e f l u o r i n e i s s m a l l e r than a h y d r o x y l i t i s c o n s i d e r a b l y l a r g e r than a hydrogen when the bond length i s taken i n t o account; a point f r e q u e n t l y overlooked i n s t u d i e s where f l u o r i n e i s used as a replacement f o r h y d r o g e n .

Table I .


GROUP

A Comparison of S i z e f o r Some F u n c t i o n a l Groups (Data from R e f e r e n c e 1) BOND LENGTH (A°)

V A N DER W A A L S

TOTAL

R A D I U S (A°)

(A°)

C-H

1.09

1.20

2.29

C-F

1.39

1.35

2.74

C-O(H)

1.43

1.40

2.83

C-OH

1.43

2.10

3.53

S e c o n d l y , the hydrogen bonding c a p a b i l i t i e s of these s u b s t i t u e n t s vary c o n s i d e r a b l y . The o r i g i n a l h y d r o x y l , when o p t i m a l l y bound w i t h i n a p r o t e i n , can be i n v o l v e d i n two hydrogen bonds as an a c c e p t o r ( p r o t o n a c c e p t o r ) and one hydrogen bond as a donor (see R e f e r e n c e 2^ f o r some b e a u t i f u l examples of such c o m p l e x e s ) . The hydrogen i n a deoxy sugar cannot be i n v o l v e d i n any s i g n i f i c a n t hydrogen b o n d i n g . However, w h i l e the f l u o r i n e i n a d e o x y f l u o r o sugar cannot p o s s i b l y donate a hydrogen bond, i t c a n , a r g u a b l y , act as a hydrogen bond acceptor (3). Thus the p o t e n t i a l f o r hydrogen bonding i s m o d i f i e d i n an i n t e r e s t i n g way w i t h i n such a s e r i e s . The e x t e n t of involvement of f l u o r i n e a t t a c h e d to c a r b o n , i n hydrogen bonding i s a matter of some d e b a t e . The h i g h e l e c t r o n e g a t i v i t y of f l u o r i n e c l e a r l y f a v o u r s bonding of t h i s t y p e , but s i n c e i t s lone p a i r e l e c t r o n s are h e l d t i g h t l y to the f l u o r i n e n u c l e u s i t might be expected t h a t the r e s u l t a n t hydrogen bonds would be somewhat weakened. E v i d e n c e f o r such hydrogen bonding i n s o l u t i o n and i n the gas phase does, however, exist. F o r example the r e l a t i v e l y h i g h b o i l i n g p o i n t of d i f l u o r o methane has been a t t r i b u t e d (4) t o i n t e r m o l e c u l a r hydrogen bonding interactions involving fluorine. A s i m i l a r e x p l a n a t i o n was p r o v i d e d f o r the o b s e r v e d d i m e r i z a t i o n of 2 , 2 , 2 - t r i f l u o r o e t h a n o l i n the gas phase ( 5 ) . B e t t e r e v i d e n c e f o r such hydrogen bonding i n t e r a c t i o n s comes from X - r a y c r y s t a l l o g r a p h i c d a t a . A r e c e n t review (6) of over 260 s t r u c t u r e s c o n t a i n i n g the C - F fragment r e v e a l e d n i n e such i n t e r a c t i o n s , the m a j o r i t y of which were C - F » » » H - N hydrogen bonds. Two o t h e r more r e c e n t s t r u c t u r e s have shown the e x i s t e n c e of C - F » » « H - 0 hydrogen bonds i n a f l u o r i n a t e d c a r b o x y l i c a c i d (7) and i n a d i f l u o r i n a t e d sugar ( 8 ) . I n the l a t t e r example (see F i g u r e 1 ) , both the f l u o r i n e s were i n v o l v e d i n weak, but s i g n i f i c a n t , i n t e r a c t i o n s as



WITHERS ET AL.

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F i g u r e 1. S t e r e o v i e w of the p a c k i n g of 2 - d e o x y - 2 - f l u o r o - ( 3 - D mannopyranosyl f l u o r i d e . Broken l i n e s r e p r e s e n t hydrogen bonds and H " « ( F / 0 ) i n t e r a c t i o n s w i t h H«"F H Gly 674 N (D)

OH-6

jT^ y

His 376 N D 1 ' A s n 483 O D 1 (A)

N© H /IH2 —C 0

mt


67



Scheme 1. G e n e r a l mechanism of a - g l u c o s y l t r a n s f e r a s e s . R may be one of a v a r i e t y of a g l y c o n e s . R'OH may r e p r e s e n t water or the n o n - r e d u c i n g terminus of an o l i g o s a c c h a r i d e .



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atom, e s p e c i a l l y d i r e c t l y a d j a c e n t to the anomeric c e n t r e , s h o u l d r e s u l t i n i n d u c t i v e d e s t a b i l i s a t i o n of such c a t i o n i c s p e c i e s or states. T h i s s h o u l d r e s u l t i n an o v e r a l l d e c r e a s e i n the t u r n o v e r rate. The c o n v e r s e s h o u l d be t r u e f o r deoxy s u g a r s , thus t h e s e s h o u l d t u r n o v e r more r a p i d l y based on t h i s c r i t e r i o n . As a t e s t f o r t h i s we have s t u d i e d a model r e a c t i o n , the a c i d c a t a l y s e d h y d r o l y s i s of a s e r i e s of d e o x y f l u o r o - D - g l u c o p y r a n o s y l phosphates. A c i d - c a t a l y s e d h y d r o l y s i s of the p a r e n t sugar phosphate was demonstrated p r e v i o u s l y to p r o c e e d v i a an oxocarbonium i o n i n t e r m e d i a t e by Bunton e t a l . ( 3 3 , 3 4 ) who a l s o determined the mechanism of bond c l e a v a g e at a v a r i e t y of pH v a l u e s . More r e c e n t work (35) a d d r e s s e d the e f f e c t s of s t r u c t u r a l changes i n the sugar on the r a t e of a c i d - c a t a l y s e d h y d r o l y s i s of i t s 1-phosphate. The r e s u l t s were seen to be c o n s i s t e n t w i t h those o b t a i n e d f o r the a c i d - c a t a l y s e d h y d r o l y s i s of e q u i v a l e n t a l k y l and a r y l glucopyranosides (36-38). We have measured the f i r s t o r d e r r a t e c o n s t a n t s f o r the a c i d c a t a l y s e d (1M HCIO^) h y d r o l y s i s of the s e r i e s of d e o x y f l u o r o - D - g l u c o p y r a n o s y l phosphates and t h e s e are g i v e n i n T a b l e I V .

Table IV.

A c i d C a t a l y s e d H y d r o l y s i s Data

Compound

Hydrolysis

Rate Constant

Temperature (°C)

ko x 105 sec-1

a-D-glucopyranosyl phosphate

25

4.10

6-deoxy-6-fluoro-a-

25

1.12

4-deoxy-4-fluoro-a-

25

0.270

3-deoxy-3-fluoro-a-

25

0.480

2-deoxy-2-fluoro-a-

25

0.068

2-deoxy-2-fluoro- /3-

25

0.175

C l e a r l y f l u o r i n e s u b s t i t u t i o n h a s , i n each c a s e , lowered the r a t e r e l a t i v e to t h a t of the p a r e n t compound, w i t h the g r e a t e s t r a t e d e p r e s s i o n ( 6 0 - f o l d ) b e i n g f o r the 2 - s u b s t i t u t e d a n a l o g u e . A more d e t a i l e d d i s c u s s i o n of t h e s e r e s u l t s i s p r e s e n t e d elsewhere (39) but i t i s i n t e r e s t i n g to note t h a t the o r d e r of r a t e s of h y d r o l y s i s i s 6 - f l u o r o > 3 - f l u o r o > 4 - f l u o r o > 2 - f l u o r o , which i s the i n v e r s e of the o r d e r o b s e r v e d f o r the a c i d - c a t a l y s e d h y d r o l y s i s of s e r i e s o f a l k y l (40) and a r y l (41) deoxy p - D - g l u c o p y r a n o s i d e s ( i . e . 2-deoxy > 4-deoxy > 3-deoxy > 6 - d e o x y ) . T h i s inverse r e l a t i o n s h i p suggests a common, p r o b a b l y e l e c t r o n i c , o r i g i n and we p r o v i d e two p o s s i b l e



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r a t i o n a l e s as f o l l o w s . C l e a r l y i n d u c t i v e e f f e c t s w i l l be i m p o r t a n t at the 2 - p o s i t i o n , but these would be expected to be a t t e n u a t e d r a p i d l y w i t h d i s t a n c e from the anomeric c e n t r e . They m i g h t , however, be s u f f i c i e n t to at l e a s t p a r t i a l l y e x p l a i n the observed o r d e r of r a t e c o n s t a n t s i f one assumes t h a t the m a j o r i t y of the p o s i t i v e charge i n the oxocarbonium i o n r e s i d e s on the r i n g oxygen. In t h a t case the 4 - s u b s t i t u e n t i s c l o s e r to the charge than i s the 3 - p o s i t i o n so i t s e f f e c t s h o u l d be g r e a t e r . However, the 6 - p o s i t i o n i s a l s o j u s t as c l o s e , yet the 6 - f l u o r o d e r i v a t i v e h y d r o l y s e s more r a p i d l y than the 3 - f l u o r o compound. Another p o s s i b l e e x p l a n a t i o n r e l a t e s t o the r e l a t i v e o r i e n t a t i o n of d i p o l e s a s s o c i a t e d w i t h C-OH and C - F bonds i n the ground s t a t e and i n the h a l f c h a i r t r a n s i t i o n s t a t e , s i n c e i n c r e a s e d a l i g n m e n t of d i p o l e s a t the t r a n s i t i o n s t a t e would be expected to r e s u l t i n r a t e d e c r e a s e s . The importance of such d i p o l a r i n t e r a c t i o n s has been amply i l l u s t r a t e d by the anomeric e f f e c t . As d e t a i l e d elsewhere ( 3 9 ) , t h e r e i s an o v e r a l l d e c r e a s e i n d i p o l a r a l i g n m e n t f o r the 3 - f l u o r o sugar i n g o i n g t o the t r a n s i t i o n s t a t e which s h o u l d i n c r e a s e i t s r a t e r e l a t i v e l y , whereas s u b s t i t u t i o n a t the 2 - and 4 - p o s i t i o n s r e s u l t s , r e s p e c t i v e l y , i n an i n c r e a s e i n a l i g n m e n t and i n no net change. The r e s u l t of t h i s would be t o d e c r e a s e the r a t e of the 2 - f l u o r o r e l a t i v e l y and to l e a v e the 4 - f l u o r o slower than the 3 - f l u o r o . T h i s c o u l d be the cause of the o r d e r i n v e r s i o n and the c o n v e r s e would c l e a r l y f o l l o w f o r the deoxyglucosides. In r e a l i t y both e f f e c t s p r o b a b l y c o n t r i b u t e . determined f o r the The a c t i v a t i o n 2 - f l u o r o analogue (AH = 113.5 k J m o l " ; *5 ) compared w i t h those (Air = 116.6 k J m o l " ; A S * = 14.9 eu) f o r the p a r e n t sugar phosphate ( 3 3 ) . S u r p r i s i n g l y , the major d i f f e r e n c e i s i n the a c t i v a t i o n e n t r o p y which s u g g e s t s a more S 2 - l i k e t r a n s i t i o n s t a t e i n v o l v i n g c o n s i d e r a b l e p r e a s s o c i a t i o n of the water to the g l y c o s i d i c c a r b o n atom. This is quite consistent with current suggestions ( 3 8 , 4 2 ) t h a t a l l s o l v o l y s e s at sugar anomeric carbons proceed t h r o u g h a p r e - a s s o c i a t i o n mechanism and a l s o w i t h the r e c e n t o b s e r v a t i o n s (43) of i n d u c t i v e enhancement of p r e - a s s o c i a t i v e p a r t i c i p a t i o n i n other displacement r e a c t i o n s . Clearly, therefore, electronic effects can be v e r y i m p o r t a n t , but the magnitude of the e l e c t r o n i c e f f e c t s o b s e r v e d i n any case may not be p r e d i c t a b l e s i n c e the t r a n s i t i o n s t a t e s t r u c t u r e can v a r y c o n s i d e r a b l y . T h i s w i l l be p a r t i c u l a r l y t r u e , and h a r d to e v a l u a t e , at an enzyme a c t i v e s i t e . 1

A S

=

e u

a

n

d

1

5

N

Enzyme S t u d i e s ;

Glycogen Phosphorylase.

We have now s t u d i e d f l u o r i n a t e d sugars as s u b s t r a t e s of a number o f g l y c o s y l t r a n s f e r a s e s ; our most complete s t u d y to date b e i n g w i t h r a b b i t muscle g l y c o g e n p h o s p h o r y l a s e . T h i s enzyme, which c a t a l y s e s the r e v e r s i b l e p h o s p h o r o l y s i s of g l y c o g e n , p r o d u c i n g g l u c o s e - 1 phosphate i s thought to o p e r a t e v i a the e l e m e n t a r y mechanism shown i n Scheme 1 (R « p h o s p h a t e , R'OH = g l y c o g e n ) . As can be s e e n , b i n d i n g of g l u c o s e - l - p h o s p h a t e i s f o l l o w e d by the a c i d - c a t a l y s e d c l e a v a g e of the anomeric l i n k a g e l i b e r a t i n g phosphate and p r o d u c i n g a c a r b o x y l a t e - s t a b i l i s e d g l u c o s y l oxocarbonium i o n i n t e r m e d i a t e , o r more l i k e l y a c o v a l e n t g l u c o s y l enzyme. D e g l u c o s y l a t i o n of the enzyme, and consequent g l u c o s y l t r a n s f e r , w i l l o c c u r upon a t t a c k of the 4 - h y d r o x y l at the n o n - r e d u c i n g t e r m i n u s of g l y c o g e n . Thus both


5.

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71


the g l u c o s y l a t i o n and the d e g l u c o s y l a t i o n of the enzyme are c o n s i d e r e d to p r o c e e d v i a oxocarbonium i o n - l i k e t r a n s i t i o n s t a t e s . I n l i g h t of t h i s proposed mechanism i t i s of i n t e r e s t to a n t i c i p a t e what would be the e f f e c t s on t u r n o v e r , of s u b s t i t u t i n g v a r i o u s sugar h y d r o x y l s by hydrogen and by f l u o r i n e . Two major e f f e c t s come to mind, namely e l e c t r o n i c e f f e c t s and b i n d i n g e f f e c t s . The p r e v i o u s study of the a c i d - c a t a l y s e d h y d r o l y s i s of the s e r i e s of d e o x y f l u o r o - and d e o x y - a - D - g l u c o s e - l - p h o s p h a t e analogues has amply demonstrated the importance of e l e c t r o n i c e f f e c t s from such s u b s t i t u e n t s on r e a c t i o n s p r o c e e d i n g v i a c a t i o n i c t r a n s i t i o n s t a t e s . As was seen i n Scheme 1, t h i s enzymic r e a c t i o n i s a l s o thought to p r o c e e d v i a oxocarbonium i o n - l i k e t r a n s i t i o n s t a t e s . On the b a s i s of electronic effects alone, therefore, the deoxy s u b s t r a t e s would be expected to t u r n over more r a p i d l y than the normal s u b s t r a t e and the d e o x y f l u o r o s u b s t r a t e s more s l o w l y . B i n d i n g e f f e c t s are somewhat more complex. As has been d i s cussed many times p r e v i o u s l y ( 1 3 , 4 4 , 4 5 ) , enzymes c a t a l y s e r e a c t i o n s by s p e c i f i c a l l y b i n d i n g the r e a c t i o n t r a n s i t i o n s t a t e s t r u c t u r e , t h e r e b y s t a b i l i s i n g i t and l o w e r i n g the o v e r a l l a c t i v a t i o n f r e e energy. Thus b i n d i n g energy i s used i n a f a i r l y d i r e c t way to e f f e c t catalysis. Some v e r y e l e g a n t s t u d i e s i n v o l v i n g p r o t e i n mutagenesis have been performed to measure the s t r e n g t h s of i n d i v i d u a l hydrogen bonds i n v o l v e d i n s t a b i l i s i n g the r e a c t i o n t r a n s i t i o n s t a t e f o r the enzyme t y r o s y l t-RNA s y n t h e t a s e ( 1 3 , 4 6 ) . These s t u d i e s s u g g e s t e d t h a t t h i s enzyme takes advantage of the g e o m e t r i c a l changes i n the s u b s t r a t e s t r u c t u r e on a p p r o a c h i n g the t r a n s i t i o n s t a t e , to s e l e c t i v e l y s t a b i l i z e the t r a n s i t i o n s t a t e , t h e r e b y f a c i l i t a t i n g the reaction. A s i m i l a r s i t u a t i o n may o b t a i n w i t h g l y c o g e n p h o s p h o r y l a s e , s i n c e the t r a n s i t i o n s t a t e oxocarbonium i o n s p e c i e s w i l l have a h a l f - c h a i r c o n f o r m a t i o n , d i s t i n c t l y d i f f e r e n t from the ground-state chair conformation. Thus i n d i v i d u a l hydrogen bonds may be v e r y i m p o r t a n t i n promoting such a t r a n s i t i o n . Any s u b s t i t u t i o n i n the sugar r i n g which e l i m i n a t e s hydrogen bonds i n v o l v e d i n t r a n s i t i o n s t a t e b i n d i n g w i l l t h e r e f o r e r e s u l t i n lower t u r n o v e r rates. On t h i s b a s i s a l o n e , t h e r e f o r e , the deoxy s u b s t r a t e s s h o u l d be the s l o w e s t , s i n c e no hydrogen bonding i s p o s s i b l e to the hydrogen substituent. The d e o x y f l u o r o s u b s t r a t e s may be as poor as the deoxy, b u t , on a v e r a g e , s h o u l d be s l i g h t l y b e t t e r due to t h e i r c a p a c i t y f o r a c c e p t i n g hydrogen bonds. These two f a c t o r s , b i n d i n g , and i n t r i n s i c e l e c t r o n i c e f f e c t s can be e x p e c t e d , t h e r e f o r e , to combine and to a f f e c t r a t e s of t u r n o v e r . U n f o r t u n a t e l y , however, i t i s not a s i m p l e t a s k to d i s s e c t t h e s e two e f f e c t s w i t h any p r e c i s i o n . Even though the i n t r i n s i c e l e c t r o n i c e f f e c t s a r e known f o r a model r e a c t i o n , the a c i d - c a t a l y s e d h y d r o l y s i s of the same compounds, t h e s e cannot be a p p l i e d d i r e c t l y to the e n z y m a t i c r e a c t i o n s i n c e the a b s o l u t e magnitude of such e l e c t r o n i c e f f e c t s depends upon the p r e c i s e t r a n s i t i o n s t a t e s t r u c t u r e . It i s v e r y u n l i k e l y t h a t these t r a n s i t i o n s t a t e s t r u c t u r e s f o r the enzymec a t a l y z e d and a c i d - c a t a l y z e d r e a c t i o n s w i l l be i d e n t i c a l . T a b l e V g i v e s the M i c h a e l i s - M e n t e n p a r a m e t e r s , V and Km, measured f o r t u r n o v e r of t h e s e s u b s t r a t e analogues by r a b b i t muscle phosphorylase b. V x/* used i n t h i s a n a l y s i s s i n c e they r e p r e s e n t the s e c o n d o r d e r r a t e f o r r e a c t i o n of f r e e enzyme and f r e e s u b s t r a t e which can be r e l a t e d to o v e r a l l a c t i v a t i o n f r e e e n e r g i e s . m

m

v

a

l

u

e

s

a

r

a

x

e

Ja3i


72



Table V .

Kinetic

Parameters f o r G l y c o g e n P h o s p h o r y l a s e b

MUSCLE PHOSPHORYLASE b COMPOUND (L«min~lmg~l)

AAG? (kcal-mol-1)

G1P

187500

-

2-FLUORO G1P

1.08

7.3

3-FLUORO G1P

2.2

6.8

4-FLUORO G1P

37

5.1

6-FLUORO G1P

0.26

8.1

2-DEOXY G1P

n.d.

n.d.

3-DEOXY G1P

5.8

6.3

4-DEOXY G1P

400

3.7

6-DEOXY G1P

11.1

5.9

Vm/Km x 10"

4

AAG^ r e p r e s e n t s the d i f f e r e n c e i n a c t i v a t i o n f r e e e n e r g i e s of c a t a l y s e d r e a c t i o n of g l u c o s e - l - p h o s p h a t e and the r e s p e c t i v e analogue. T h i s i s c a l c u l a t e d from E q u a t i o n 3 where (y^x^^i v a l u e f o r g l u c o s e - l - p h o s p h a t e and ( / )2 analogue. V

K m

i

s

t

n

a

t

f

o

r

t

n

e

m a x


enzymeIs

the

5.

WITHERS ETAL.


73

C l e a r l y they a r e a l l v e r y slow s u b s t r a t e s which r e a c t at r a t e s some 10 t o 10 times slower than the normal s u b s t r a t e . D e s p i t e the a f o r e m e n t i o n e d d i f f i c u l t i e s i n d i s s e c t i n g the e l e c t r o n i c and b i n d i n g f a c t o r s s e v e r a l i m p o r t a n t q u e s t i o n s can be a d d r e s s e d as f o l l o w s : (a) does e n z y m a t i c c a t a l y s i s p r o c e e d v i a an oxocarbonium i o n t r a n s i t i o n state? I t was proposed e a r l i e r t h a t , on the b a s i s of b i n d i n g f a c t o r s o n l y , the deoxy sugars s h o u l d g e n e r a l l y be worse s u b s t r a t e s than the d e o x y f l u o r o s u g a r s . However, the c o n v e r s e i s c l e a r l y seen to be t r u e i n comparing V / K m values. T h i s can o n l y be e x p l a i n e d i f the i n t r i n s i c e l e c t r o n i c e f f e c t s a r e i m p o r t a n t and the t r a n s i t i o n s t a t e has s u b s t a n t i a l oxonium i o n c h a r a c t e r . These d a t a t h e r e f o r e p r o v i d e s u b s t a n t i a l s u p p o r t f o r the mechanism proposed, (b) How s t r o n g a r e the hydrogen bonds at the t r a n s i t i o n s t a t e ? A minimum e s t i m a t e of such hydrogen bond s t r e n g t h s can be o b t a i n e d by c o n s i d e r i n g V ^ ^ K m v a l u e s f o r the d e o x y - s u g a r s e r i e s . S i n c e i n t r i n s i c e l e c t r o n i c e f f e c t s w i l l tend to speed up t h e i r t u r n o v e r , then any r a t e r e d u c t i o n observed must be due e n t i r e l y to p o o r e r b i n d i n g at: the t r a n s i t i o n s t a t e . The i n c r e a s e s i n a c t i v a t i o n f r e e e n e r g y , AAG* can be r e a d i l y c a l c u l a t e d from E q u a t i o n 3. These v a l u e s are l i s t e d i n T a b l e V and a r e minimum e s t i m a t e s of the s t r e n g t h s of hydrogen bonds to the


m a x

M G * = RT i n W * » > 2 (

(3)

< W * « > i c o r r e s p o n d i n g h y d r o x y l a t the t r a n s i t i o n s t a t e . The v a l u e s o b t a i n e d a r e c o n s i d e r a b l y l a r g e r than those measured i n the study of g l u c o s e b i n d i n g to the T - s t a t e enzyme. This is consistent with expectations r e g a r d i n g i n c r e a s e d hydrogen bond s t r e n g t h s at the t r a n s i t i o n s t a t e . F u r t h e r , i t i s c l e a r t h a t the hydrogen bonds at the 3- and 6p o s i t i o n s a r e the s t r o n g e s t , as was observed f o r g l u c o s e b i n d i n g to T - s t a t e enzymes. T h i s s u g g e s t s a common g l u c o s e s u b - s i t e w h i c h remains i n t a c t d u r i n g the T*R t r a n s i t i o n . A Mechanism-Based I n h i b i t o r f o r a g - G l u c o s i d a s e . Glucosidasec a t a l y s e d h y d r o l y s e s are g e n e r a l l y c o n s i d e r e d to proceed v i a a "lysozyme type" of mechanism s i m i l a r to t h a t d i s c u s s e d p r e v i o u s l y f o r g l y c o g e n p h o s p h o r y l a s e and i l l u s t r a t e d i n Scheme 2 f o r a " r e t a i n i n g " glucosidase. Thus g l u c o s i d e h y d r o l y s i s proceeds through some form of g l u c o s y l - e n z y m e i n t e r m e d i a t e and v i a a t r a n s i t i o n s t a t e w i t h s u b s t a n t i a l oxocarbonium i o n c h a r a c t e r . On the b a s i s of our p r e v i o u s s t u d i e s we reasoned t h a t s u b s t i t u t i o n of the 2 - h y d r o x y l by an e l e c t r o n e g a t i v e f l u o r i n e s h o u l d d e s t a b i l i s e the t r a n s i t i o n s t a t e s f o r g l u c o s y l a t i o n (k^) and d e g l u c o s y l a t i o n (k2) of the enzyme, thus s l o w i n g down both these s t e p s . However, f u r t h e r i n c o r p o r a t i o n of a r e a c t i v e l e a v i n g group as the a g l y c o n e i n t o such d e a c t i v a t e d s u b s t r a t e s might i n c r e a s e the g l u c o s y l a t i o n r a t e s u f f i c i e n t l y ( w i t h o u t a f f e c t i n g the d e g l u c o s y l a t i o n r a t e ) to p e r m i t t r a p p i n g o f the 2 - d e o x y - 2 - f l u o r o - D - g l u c o s y l enzyme i n t e r m e d i a t e . I f the d e g l u c o s y l a t i o n r a t e i s s u f f i c i e n t l y slow t h i s might p e r m i t i s o l a t i o n of an i n a c t i v a t e d enzyme. Thus, 2 , 4 - d i n i t r o p h e n y l 2-deoxy-2-fluoro-0-D-glucopyranoside s y n t h e s i z e d by d e p r o t e c t i o n of i t s t r i - O - a c e t a t e , which had been


was


74


Scheme 2. G e n e r a l mechanism f o r 3 - g l u c o s i d a s e s r e a c t i o n w i t h r e t e n t i o n of c o n f i g u r a t i o n .

catalysing

p r e p a r e d by t r e a t m e n t of 2 , 3 , 6 - t r i - 0 - a c e t y l - 2 - d e o x y - 2 - f l u o r o - D g l u c o p y r a n o s e w i t h f l u o r o d i n i t r o b e n z e n e , i n the presence of base (l,4-diazabicyclo[2.2.2]octane) (47). I n c u b a t i o n of A l c a l i g e n e s f a e c a l i s p - g l u c o s i d a s e (48) w i t h t h i s compound r e s u l t e d i n a r a p i d , t i m e - d e p e n d e n t l o s s of enzyme a c t i v i t y . T h i s f o l l o w e d pseudof i r s t - o r d e r k i n e t i c s as shown i n F i g u r e 2, p e r m i t t i n g e s t i m a t i o n of a d i s s o c a t i o n c o n s t a n t (K^) of 0.05 mM and an i n a c t i v a t i o n r a t e c o n s t a n t (k^) of 25 minute . The c o m p e t i t i v e i n h i b i t o r i s o p r o p y l t h i o 3 - D - g l u c o p y r a n o s i d e p r o v i d e d p r o t e c t i o n a g a i n s t i n a c t i v a t i o n as shown i n F i g u r e 2, p r o v i d i n g e v i d e n c e t h a t i n a c t i v a t i o n i s due to r e a c t i o n at the a c t i v e s i t e .



5.

WITHERS ET AL.

75


F i g u r e 2. I n a c t i v a t i o n of A . f a e c a l i s g - g l u c o s i d a s e w i t h 2 , 4 dinitrophenyl 2-deoxy-2-fluoro 3-D-glucopyranoside. a) I n a c t i v a t i o n at the f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s ( O 0.5 uM; • = 1.0 uH; • = 2.0uM; • = 3.0 uM; A = 4 . 0 uM; = 5.0 uM). b) P r o t e c t i o n a g a i n s t i n a c t i v a t i o n g i v e n by i s o p r o p y l 3-D-thioglucopyranoside. VQ i s the i n i t i a l enzyme a c t i v i t y , V i s the o b s e r v e d enzyme a c t i v i t y at the times i n d i c a t e d . Enzyme a c t i v i t y was measured by s p e c t r o p h o t o m e t r y a s s a y of p - n i t r o p h e n o l r e l e a s e from p - n i t r o p h e n y l - p - D - g l u c o p y r a n o s i d e i n 50 mM sodium phosphate b u f f e r , pH 6 . 8 . A


76


T h i s t h e r e f o r e r e p r e s e n t s a n o v e l type of mechanism-based i n h i b i t o r of g l y c o s i d a s e s which s h o u l d be f a i r l y g e n e r a l l y a p p l i c a b l e to t h i s c l a s s of enzymes, p r o v i d e d t h a t the d e g l y c o s y l a t i o n r a t e f o r the analogue i s s u f f i c i e n t l y slow r e l a t i v e to the g l y c o s y l a t i o n r a t e . E x p e r i m e n t s are c u r r e n t l y planned to i d e n t i f y the a c t i v e s i t e n u c l e o p h i l e i n t h i s enzyme u s i n g such an i n h i b i t o r and to c o n f i r m i t s mode of a c t i o n by p u r i f y i n g the i n a c t i v e d e o x y f l u o r o g l u c o s y l enzyme and s t u d y i n g i t by F NMR. The g e n e r a l a p p l i c a b i l i t y of t h i s a p p r o a c h i s a l s o b e i n g t e s t e d w i t h a v a r i e t y of g l y c o s i d a s e s by use of the a p p r o p r i a t e 2 - d e o x y - 2 - f l u o r o - g l y c o s i d e i n each c a s e .


1

9

Conclusions S u i t a b l e a p p l i c a t i o n of deoxy- and d e o x y f l u o r o - analogues of e n z y m a t i c s u b s t r a t e s has p r o v i d e d v a l u a b l e i n s i g h t i n t o the mechanisms of e n z y m e - c a t a l y s e d g l u c o s y l t r a n s f e r and i n t o the r o l e of hydrogen bonding i n s u b s t r a t e r e c o g n i t i o n and c a t a l y s i s . I t has a l s o p e r m i t t e d d e s i g n of a n o v e l c l a s s of mechanism-based enzyme inhibitors. A n o t h e r a p p e a l i n g a p p l i c a t i o n of such compounds i s t h e i r use i n F nmr s t u d i e s of e n z y m e - s u b s t r a t e complexes. We have r e c e n t l y i n i t i a t e d such s t u d i e s on the enzyme phosphoglucomutase. Acknowledgments T h i s work was s u p p o r t e d by g r a n t s from the N a t u r a l S c i e n c e s and E n g i n e e r i n g R e s e a r c h C o u n c i l of Canada and from the B r i t i s h Columbia H e a l t h Care R e s e a r c h F o u n d a t i o n .

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11

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RECEIVED March 14, 1988


Fluorinated Carbohydrates as Probes of Enzyme Specificity and

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