Chapter 43 β-Glucosidases:
M e c h a n i s m and Inhibition
Stephen G. Withers and Ian P. Street Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Y6, Canada
The generally accepted mechanism of action of glycosi dases which hydrolyse glycosides with overall retention of configuration at the anomeric center involves a dou ble displacement. Initial general acid-catalyzed gener ation of a glycosyl-enzyme intermediate is followed by its general base-catalyzed hydrolysis. Both the forma tion and the hydrolysis of the glycosyl-enzyme can be considered to proceed via oxocarbonium ion-like transi tion states. Destabilization of such transition states can be achieved by replacing the C-2 hydroxyl of the sub strate by the more electronegative fluorine, thus slow ing both steps. Simultaneous incorporation of an ex cellent leaving group (fluoride or dinitrophenolate) as the aglycone permits the accumulation of the interme diate which is sufficiently stable to be isolated. In vestigation of such an intermediate generated on a β -glucosidase, by means of F-NMR, allowed its iden tification as an α-D-glucopyranosyl-enzyme. Activated 2-deoxy-2-fluoroglycosides therefore act as mechanism -based inactivators, thereby representing a new class of "suicide" inactivators for glycosidases. 19
/?-Glucosidases p l a y a n i m p o r t a n t role i n t h e d e g r a d a t i o n o f cellulose b y h y d r o l y z i n g cellobiose t o glucose. I n this way, n o t o n l y is t h e key m e t a b o l i t e glucose p r o d u c e d , b u t also cellobiose, a n i n h i b i t o r o f exoglucanases, is removed. A n u n d e r s t a n d i n g o f the detailed c h e m i c a l m e c h a n i s m o f a c t i o n of t h i s class o f enzymes is therefore i m p o r t a n t b o t h i n terms o f possible c h e m i c a l , o r genetic engineering approaches t o generation o f a r t i f i c i a l e n zymes a n d also i n t h e design o f specific i n h i b i t o r s w h i c h c o u l d be v a l u a b l e i n p r e v e n t i n g cellulose d e g r a d a t i o n .
0097-6156/89/0399-0597$06.00/0 © 1989 American Chemical Society
598
PLANT C E L L W A L L P O L Y M E R S
A l l glycosidases s t u d i e d to date have been f o u n d to effect h y d r o l y s i s of the g l y c o s i d i c linkage b y cleavage of the b o n d between the a n o m e r i c c a r b o n a n d the g l y c o s i d i c oxygen. However, two different stereochemical o u t c o m e s of such a h y d r o l y t i c m e c h a n i s m are possible. T h e b o n d can be cleaved w i t h r e t e n t i o n of a n o m e r i c c o n f i g u r a t i o n (by a " r e t a i n i n g " glycosidase) (1-3), or w i t h i n v e r s i o n of c o n f i g u r a t i o n (by a n " i n v e r t i n g " glycosidase) (1-3). M a n y e x a m p l e s of each t y p e have been f o u n d . P r o b a b l y the m o s t c o m m o n class, a n d c e r t a i n l y the best defined m e c h a n i s t i c a l l y , is t h a t of the " r e t a i n i n g " glycosidases. T h e most generally accepted m e c h a n i s m of a c t i o n for such enzymes is s h o w n i n F i g u r e 1 for a " r e t a i n i n g " /?-glucosidase, a n d involves a n active site c o n t a i n i n g two m e c h a n i s t i c a l l y i m p o r t a n t residues; a n a c i d c a t a l y s t whose i d e n t i t y i n different systems is thought to be a c a r b o x y l g r o u p or t y r o s i n e , a n d a nucleophile considered to be a c a r b o x y l a t e residue i n essentially a l l cases (see references 1-3 for reviews of glycosidase m e c h a n i s m s ) . B i n d i n g of the glycoside substrate is t h o u g h t to be followed b y p r o t o n d o n a t i o n f r o m the a c i d c a t a l y t i c group to the glycosidic oxygen a t o m , thereby increasing the l a b i l i t y of the g l y c o s i d i c b o n d . B o n d heterolysis e n sues, generating a free (aglyconic) a l c o h o l w h i c h departs, a n d a g l y c o s y l o x o c a r b o n i u m i o n species s t a b i l i z e d by i n t e r a c t i o n w i t h the c a r b o x y l a t e g r o u p . T h e t i m i n g o f t h i s " a t t a c k " of the c a r b o x y l a t e g r o u p is p r o b a b l y such t h a t the r e a c t i o n has considerable Sjv2 character w i t h no true o x o c a r b o n i u m i o n i n t e r m e d i a t e , b u t r a t h e r j u s t a t r a n s i t i o n state w i t h s u b s t a n t i a l o x o c a r b o n i u m i o n character. S u c h " p r e - a s s o c i a t i o n " of the nucleophile has also been suggested to be very c o m m o n i n n o n - e n z y m i c g l y c o s y l transfer reactions (4). T h u s the first step of the p a t h w a y involves the a c i d - c a t a l y z e d f o r m a t i o n of a g l y c o s y l - e n z y m e i n t e r m e d i a t e v i a a n o x o c a r b o n i u m i o n - l i k e t r a n s i t i o n state. C o m p l e t i o n o f the process involves a t t a c k of water at the a n o m e r i c center, w i t h some general base c a t a l y t i c assistance g e n e r a t i n g , i n t h i s case, /?-D-glucopyranose as p r o d u c t . T h i s second g l y c o s y l transfer step, d e g l y c o s y l a t i o n , w i l l p r e s u m a b l y also occur v i a a t r a n s i t i o n state w i t h s u b s t a n t i a l o x o c a r b o n i u m i o n character. A l t e r n a t i v e s to t h i s m e c h a n i s m , w h i c h differ i n some cases o n l y r e l a t i v e l y s u b t l y , b u t i n other cases quite d r a m a t i c a l l y , are not w e l l s u p p o r t e d , b u t i n c l u d e a possible m e c h a n i s m i n v o l v i n g a n i n i t i a l e n d o c y c l i c C - 0 b o n d cleavage (5), a n d one i n v o l v i n g a f u l l o x o c a r b o n i u m i o n i n t e r m e d i a t e . T h e case against such mechanisms is discussed i n some d e t a i l elsewhere (2). T h e work described i n this paper s u m m a r i z e s our recent a t t e m p t s to p r o v i d e s u b s t a n t i a l proof for the m e c h a n i s m of F i g u r e 1. T h i s was achieved b y t r a p p i n g a n d i s o l a t i n g the g l y c o s y l - e n z y m e i n t e r m e d i a t e , thus p r o v i n g its existence, a n d also by d i r e c t l y d e t e r m i n i n g the stereochemistry of the linkage of this sugar to the e n z y m e . S u c h studies, b y t h e i r very n a t u r e , led to the generation of a new class of mechanism-based i n a c t i v a t o r s of glycosidases. Results and Discussion Since b o t h f o r m a t i o n a n d h y d r o l y s i s of the g l y c o s y l - e n z y m e proceed v i a o x o c a r b o n i u m i o n - l i k e t r a n s i t i o n states, i t seemed reasonable to assume,
43.
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599
F i g u r e 1. M e c h a n i s m of a " r e t a i n i n g " /?-glucosidase. O R = the aglycone; N u = the e n z y m e ' s n u c l e o p h i l e ; H A = the e n z y m e ' s a c i d c a t a l y s t .
600
PLANT C E L L W A L L POLYMERS
i n l i n e w i t h our m o d e l n o n - e n z y m i c studies (6), t h a t s u b s t i t u t i o n of the sugar h y d r o x y l at C - 2 b y the more electronegative fluorine w o u l d result i n significant i n d u c t i v e d e s t a b i l i z a t i o n of the adjacent p o s i t i v e charge at the t r a n s i t i o n state. T h i s s h o u l d result i n decreased rates of g l y c o s y l - e n z y m e f o r m a t i o n and h y d r o l y s i s , thereby p r o d u c i n g very slow substrates. S u c h i n deed was observed to be the case. However, the i n c o r p o r a t i o n of a r e l a t i v e l y reactive l e a v i n g group ( d i n i t r o p h e n o l a t e or fluoride) as the aglycone i n t o such deactivated substrates m i g h t accelerate the rate of g l y c o s y l - e n z y m e f o r m a t i o n sufficiently, without affecting the rate of g l y c o s y l - e n z y m e h y d r o l ysis, to p e r m i t a c c u m u l a t i o n a n d t r a p p i n g of the 2-deoxy-2-fluoro-glycosyle n z y m e i n t e r m e d i a t e . T h i s c o u l d allow the s t r u c t u r e a n d properties of such an i n t e r m e d i a t e to be i n v e s t i g a t e d . Inactivation Studies. T h i s strategy was tested i n i t i a l l y o n a /?-glucosidase isolated f r o m Alcaligenes faecalis (7,8) a n d since cloned a n d expressed at h i g h levels i n E. coli (9), henceforward referred to as p A B G 5 /?-glucosidase. I n the i n i t i a l k i n e t i c c h a r a c t e r i z a t i o n (8), i t h a d been noted t h a t the best substrates (highest V x and V / K ) i n c l u d e d 2 , 4 - d i n i t r o p h e n y l β-Όglucopyranoside a n d / 3 - D - g l u c o s y l fluoride. Therefore the most p r o m i s i n g candidates for t r a p p i n g of a g l y c o s y l - e n z y m e i n t e r m e d i a t e appeared to be 2 , 4 - d i n i t r o p h e n 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 i d e ( 2 F / ? D N P G l u ) a n d 2-deoxy-2-fluoro-/?-D-glucopyranosyl fluoride ( 2 F / ? G l u F ) . These two c o m p o u n d s were synthesized as described p r e v i o u s l y (10,11,12,13) a n d tested w i t h p A B G 5 /?-glucosidase for a c c u m u l a t i o n of a n i n t e r m e d i a t e . S u c h testing was r e l a t i v e l y facile, as the a c c u m u l a t i o n of the i n t e r m e d i a t e resulted i n a n apparent time-dependent i n a c t i v a t i o n of the e n z y m e . T h i s o c c u r r e d because the free enzyme, capable of i n t e r a c t i n g w i t h s u b s t r a t e , was converted i n t o the r e l a t i v e l y inert 2-fluoroglucosyl-enzyme i n t e r m e d i ate. B o t h c o m p o u n d s were indeed f o u n d to be excellent t i m e - d e p e n d e n t i n a c t i v a t o r s (10,14), r e a c t i n g according to the expected pseudo-first-order k i n e t i c s , as s h o w n for 2 F / ? D N P G l u i n F i g u r e 2. T h e rate of i n a c t i v a t i o n was dependent u p o n the c o n c e n t r a t i o n of i n a c t i v a t o r , s h o w i n g s a t u r a t i o n k i n e t i c s as expected for a s i m p l e i n a c t i v a t o r b i n d i n g n o n - c o v a l e n t l y i n i t i a l l y w i t h a dissociation constant, a n d t h e n i n a c t i v a t i n g the enzyme w i t h a rate constant k{ a c c o r d i n g to the scheme below. ma
m
Ki Ε +
a
a
:
m
k Ιτ±Ε.Ι-=±Ε-Ι
T h e f o l l o w i n g constants were d e t e r m i n e d : 2 F / ? D N P G l u , ki m i n " , Ki = 0.05 m M ; 2 F / ? G l u F , ki = 5.9 m i n " , Α\· = 0.4 m M . 1
=
25
1
Evidence for the Proposed Mechanism. A considerable effort was then ex pended i n p r o v i n g the mode of i n a c t i v a t i o n . E v i d e n c e t h a t i n a c t i v a t i o n o c c u r r e d by b i n d i n g at the active site was given by the observation t h a t 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 - / ? - D - g l u c o p y r a n o s i d e ( I P T G ) (Ki - 4 m M ) p r o v i d e d the expected p r o t e c t i o n against i n a c t i v a t i o n as s h o w n i n F i g u r e 2c. I n a c t i v a t i o n of the enzyme was a c c o m p a n i e d by the release of a " b u r s t " of aglycone ( d i n i t r o p h e n o l a t e or fluoride) as required b y such a
43.
WITHERS & STREET
ι 1/S
β-Glucosidoses: Mechanism & Inhibition
2 (μΜ-1)
·
ι
Time
(min)
601
*
F i g u r e 2. I n a c t i v a t i o n of p A B G 5 /?-glucosidase w i t h 2 F / ? D N P G l u ( s t r u c t u r e s h o w n ) (a) /?-glucosidase i n c u b a t e d w i t h the f o l l o w i n g c o n c e n t r a t i o n s of 2 F / ? D N P G l u a n d aliquots assayed against p - n i t r o p h e n y l /?-glucopyranoside at the times s h o w n : Ο = 0.5μΜ; CZ| = 1-ΟμΜ, φ = 2.0μΜ; • = 3 . 0 / i M ; A = 4 . 0 / i M ; Δ = 5 . 0 / i M ) . (b) R e p l o t of first-order rate c o n stants f r o m 2a. (c) P r o t e c t i o n against i n h i b i t i o n given b y i s o p r o p y l t h i o /?-D-glucopyranoside ( I P T G ) .
602
PLANT C E L L W A L L P O L Y M E R S
m e c h a n i s m . T h e " b u r s t " of d i n i t r o p h e n o l a t e was measured s p e c t r o p h o t o m e t r i c a l l y ( F i g u r e 3), w h i l e the " b u r s t " of fluoride was measured b y b o t h F - N M R a n d b y a d y e - b i n d i n g assay (15). In b o t h cases an essentially s t o i c h i o m e t r i c (0.93-1.0:1) reaction was observed w i t h release of one m o l e o f aglycone per mole of enzyme i n a c t i v a t e d . In a d d i t i o n to h e l p i n g prove the m o d e of a c t i o n of these i n a c t i v a t o r s t h i s procedure provides a very c o n venient a n d accurate way of m e a s u r i n g the c o n c e n t r a t i o n o f active e n z y m e present at any t i m e , s i m p l y b y p e r f o r m i n g a n " a c t i v e site t i t r a t i o n " u s i n g 2F/?DNPGlu. T h e f o r m a t i o n of a 2-fluoroglucosyl-enzyme was d e m o n s t r a t e d b y means of F - N M R . A d d i t i o n of 2 F / ? G l u F ( 1 . 0 5 m M ) to a concentrated s o l u t i o n of p A B G 5 /?-glucosidase ( 0 . 7 3 m M ) i n 5 0 m M s o d i u m p h o s p h a t e buffer, p H 6.8 i n a 5 m m N M R t u b e , i n a c t i v a t e d the e n z y m e very r a p i d l y . T h e F - N M R s p e c t r u m of t h i s s a m p l e is s h o w n i n F i g u r e 4. P e a k s were assigned as follows: T h e peak at 121.4 p p m was due to i n o r g a n i c fluoride re leased u p o n i n a c t i v a t i o n o f the e n z y m e . P e a k s at 144.8 a n d 203.4 p p m were due t o F - l a n d F - 2 , respectively, of the s m a l l excess of u n r e a c t e d 2 F / ? G l u F r e m a i n i n g . T h e r e l a t i v e l y b r o a d peak at 197.3 p p m , Δ ι / = 1 3 0 H z , was due to the 2-fluoroglucosyl-enzyme f o r m e d , b o t h the c h e m i c a l shift a n d the l i n e w i d t h b e i n g consistent w i t h t h i s assignment. D i a l y s i s of such a s a m p l e resulted i n removal o f a l l signals except the b r o a d resonance at 197.3 p p m , d e m o n s t r a t i n g t h a t the sugar was indeed covalently l i n k e d . 1 9
1 9
1 9
Stereochemistry of the Intermediate. These e x p e r i m e n t s therefore d e m o n s t r a t e d t h a t a 2-fluoroglucosyl-enzyme h a d been t r a p p e d . It was t h e n of interest to prove the stereochemistry of the linkage of t h i s sugar residue to the e n z y m e . U n f o r t u n a t e l y , the F - N M R s p e c t r u m s h o w n i n F i g u r e 4 was of no use i n this r e g a r d , since the F - c h e m i c a l shift of 2-deoxy2-fluoro-glucose a n d its derivatives is relatively insensitive to the config u r a t i o n of the a n o m e r i c s u b s t i t u e n t (o> a n d /?-2-deoxy-2-fluoro-D-glucose differ i n F c h e m i c a l shift b y o n l y 0.18 p p m a n d t h e i r tetra-0-acetates b y o n l y 1.4 p p m ) . C o u p l i n g constants are not m u c h more sensitive a n d w o u l d , i n any case, be lost i n the large n a t u r a l l i n e w i d t h of the reso nance. However, the chemical shifts of 2-deoxy-2-fluoro-D-mannose a n d its derivatives were s h o w n p r e v i o u s l y to be very sensitive to a n o m e r i c c o n figuration (16), e.g., o> a n d /?-2-deoxy-2-fluoro-D-mannose differ i n F c h e m i c a l shift b y some 18.5 p p m . Since i t h a d been s h o w n p r e v i o u s l y (8) t h a t p A B G 5 /?-glucosidase e x h i b i t s considerable /?-mannosidase a c t i v i t y , it seemed t h a t 2-deoxy-2-fluoro-/?-mannosyl fluoride ( 2 F / ? M a n F ) m i g h t be a g o o d i n a c t i v a t o r of the enzyme. If so, this w o u l d allow the generation of a 2 - f l u o r o - m a n n o s y l enzyme w h i c h c o u l d be investigated b y F - N M R . F i g ure 5 shows the result of such an i n v e s t i g a t i o n where 2 F / ? M a n F ( 1 . 5 2 m M ) has been added to p A B G 5 /?-glucosidase ( 0 . 7 4 m M ) i n 5 0 m M s o d i u m phos p h a t e buffer, p H 6.8, a n d placed i n a 5 m M N M R t u b e . T h e resonance at 121.0 p p m was due to inorganic fluoride released u p o n i n a c t i v a t i o n , whereas resonances at 149.5 a n d 224.4 p p m corresponded to F - l a n d F - 2 , respec tively, of the excess 2 F / ? M a n F e m p l o y e d . T h e b r o a d peak at 201.0 p p m was due to the 2-fluoro-mannosyl-enzyme a d d u c t . T h e c h e m i c a l shift o b 1 9
1 9
1 9
1 9
1 9
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β-Glucosidases: Mechanism & Inhibition
603
2A4initroph«nol (nmole) F i g u r e 3. M e a s u r e m e n t of the " b u r s t " of d i n i t r o p h e n o l a t e released o n re a c t i o n of 2 F / ? D N P G l u w i t h p A B G 5 /?-glucosidase. P l o t of q u a n t i t y of 2 , 4 - d i n i t r o p h e n o l released ( f r o m absorbance at 4 0 0 n m ) versus q u a n t i t y of e n z y m e t r e a t e d . S o l u t i o n s of different c o n c e n t r a t i o n s of /?-glucosidase i n 5 0 m M s o d i u m phosphate buffer, p H 6.8 were i n c u b a t e d at 3 7 ° C i n a 1 c m p a t h l e n g t h glass cuvette i n a s p e c t r o p h o t o m e t e r a n d the absorbance r e a d i n g zeroed. A s o l u t i o n of 2 F / ? D N P G l u (sufficient to p r o v i d e twice the e s t i m a t e d e n z y m e c o n c e n t r a t i o n ) was added a n d the o p t i c a l d e n s i t y at 4 0 0 n m r e c o r d e d . T h e q u a n t i t y of d i n i t r o p h e n o l released was e s t i m a t e d u s i n g a n e x t i n c t i o n coefficient of l l , 3 0 0 M c m . T h e q u a n t i t y of e n z y m e was e s t i m a t e d u s i n g a n e x t i n c t i o n coefficient at 2 8 0 n m of E° = 2 . 2 0 c m " , itself determined by a quantitative amino acid analysis. _ 1
- 1
1
%
1
604
PLANT C E L L W A L L
POLYMERS
121.4
120
140 (δ
160 ppm)
180
200
F i g u r e 4. P r o t o n decoupled F - N M R s p e c t r u m of p A B G 5 /?-glucosidase i n a c t i v a t e d w i t h 2 F / ? G l u F (conditions as described i n t e x t ) . T h i s s p e c t r u m was recorded on a 270 M H z B r u k e r / N i c o l e t i n s t r u m e n t using gated p r o t o n d e c o u p l i n g (decoupler on d u r i n g a c q u i s i t i o n o n l y ) a n d a 90° pulse angle w i t h a r e p e t i t i o n delay of 2s. A spectral w i d t h of 40,000 H z was employed a n d s i g n a l accumulated over 10,000 transients. 1 9
43.
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WITHERS & STREET
Mechanism
& Inhibition
605
149.5 224.4
201.0
120
140
206.2 224.5 DENATURED ©CYME
F i g u r e 5. P r o t o n decoupled F - N M R s p e c t r u m of p A B G 5 /?-glucosidase i n a c t i v a t e d w i t h 2 F / ? M a n F (conditions as described i n t e x t ) . S p e c t r a were recorded on a 270 M H z B r u k e r / N i c o l e t i n s t r u m e n t u s i n g gated p r o t o n de c o u p l i n g (decoupler on d u r i n g a c q u i s i t i o n o n l y ) a n d a 90° pulse angle w i t h a r e p e t i t i o n delay of 2s. A s p e c t r a l w i d t h of 40,000 H z was e m p l o y e d a n d s i g n a l a c c u m u l a t e d over 10,000 transients for the n a t i v e p r o t e i n a n d 30,000 transients for the d e n a t u r e d p r o t e i n i n 8 M u r e a , (a) F u l l s p e c t r u m w i t h e x p a n s i o n below i t ; (b) E x p a n s i o n of s p e c t r u m of d e n a t u r e d / d i a l y z e d e n zyme. 1 9
606
PLANT C E L L W A L L P O L Y M E R S
served was w e l l w i t h i n the range expected for a n α-linked 2-fluoro-mannose residue a n d some 16-20 p p m downfield of the region expected for a βlinkage. In light of the large c h e m i c a l shift differences concerned, i t was u n l i k e l y t h a t other factors affected the c h e m i c a l shift significantly. H o w ever, as described below, other factors were also considered. Since the F - N M R c h e m i c a l shifts of 2-deoxy-2-fluoro-glycosides a n d - g l y c o s y l es ters are r e l a t i v e l y insensitive to the c h e m i c a l identities of the a n o m e r i c s u b s t i t u e n t , i t was u n l i k e l y t h a t a large chemical shift resulted f r o m the replacement of the fluorine s u b s t i t u e n t at C - l by the e n z y m e n u c l e o p h i l e so t h i s was u n l i k e l y t o be the source of the large shift, especially as no s u c h large shift h a d been observed for the 2-fluoro-glucosyl enzyme. T h e b i n d i n g of fluorinated ligands to macromolecules generally produces d o w n f i e l d shifts (17) w h i c h m i g h t confuse this i n t e r p r e t a t i o n . However, since such shifts are generally s m a l l , a n d because the c h e m i c a l shift of the 2-deoxy-2fluoro-glucosyl-enzyme was i n the expected region, t h i s seemed a n u n l i k e l y cause for such a large shift. However, i n order to be c e r t a i n t h a t t h i s was not the case, the 2-fluoro-mannosyl-enzyme a d d u c t was d i a l y z e d overnight against 8 M u r e a to denature i t , a n d the F - N M R s p e c t r u m of the r e s u l t a n t unfolded p r o t e i n , i n w h i c h the fluoromannose residue w o u l d be exposed to solvent, was o b t a i n e d (see F i g u r e 5b). W h i l e a very s m a l l (A6 = 1.6 p p m ) upfield shift of the resonance was observed, the resonance r e m a i n e d well w i t h i n the region a n t i c i p a t e d for the α-linked sugar. It is of interest to note t h a t resonances a r i s i n g f r o m excess 2 F / ? M a n F were removed b y the d i a l y s i s , b u t the h i g h m o l e c u l a r weight 2-fluoromannosyl-enzyme species was r e t a i n e d . Resonances at 206.2 a n d 224.5 p p m arose f r o m a - a n d β-2deoxy-2-fluoro-D-mannose w h i c h h a d h y d r o l y z e d after d e n a t u r a t i o n of the enzyme. 1 9
1 9
Conclusion B y u s i n g 2-deoxy-2-fluoro-glycosides w i t h g o o d l e a v i n g groups at the a n o m e r i c center i t was possible to t r a p the g l y c o s y l - e n z y m e a d d u c t l o n g since p o s t u l a t e d as a n i n t e r m e d i a t e i n glycosidase c a t a l y s i s . F u r t h e r , it was possible to u n e q u i v o c a l l y prove the stereochemistry of this species. T h i s therefore provides very s t r o n g s u p p o r t i v e evidence for the m e c h a n i s m of e n z y m e - c a t a l y z e d glycoside hydrolysis s h o w n i n F i g u r e 1. T h e m e c h a n i s m involves a covalent g l y c o s y l enzyme intermediate w h i c h is formed a n d h y d r o l y z e d via o x o c a r b o n i u m i o n - l i k e t r a n s i t i o n states. T h e 2-deoxy-2fluoro-glycosides used i n this work serve as g o o d , specific mechanism-based i n a c t i v a t o r s of glycosidases, a n d m a y have future a p p l i c a t i o n i n studies of oligosaccharide a n d polysaccharide d e g r a d a t i o n . Acknowledgments W e w i s h to t h a n k D . D o l p h i n ( D e p a r t m e n t of C h e m i s t r y , U B C ) a n d R . A . J . W a r r e n a n d W . W . W a k a r c h u k ( D e p a r t m e n t of M i c r o b i o l o g y , U B C ) for their assistance i n this work.
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We also thank the Natural Sciences and Engineering Research Council of Canada and the British Columbia Health Care Research Foundation for financial support. Literature Cited 1. Sinnott, M . L. In The Chemistry of Enzyme Action; Elsevier: New York, 1984; p. 389. 2. Sinnott, M . L. In Enzyme Mechanisms; Royal Society of Chemistry, 1987; p. 259. 3. Lalegerie, P.; Legler, G.; Yon, J . M . Biochimie 1982, 64, 977. 4. Jencks, W. P. Chem. Soc. Rev. 1981, 10, 345. 5. Post, C. B.; Karplus, M . J. Amer. Chem. Soc. 1986, 108, 1317. 6. Withers, S. G.; MacLennan, D. J.; Street, I. P. Carbohydr. Res. 1986, 154, 127. 7. Han, Y . W.; Srinivasan, V. R. J. Bacteriol. 1969, 100, 1355. 8. Day, A. G.; Withers, S. G. Biochem. Cell Biol. 1986, 64, 914. 9. Wakarchuk, W. W.; Kilburn, D. G.; Miller, R. C.; Warren, R. A. J . Mol. Gen. Genet. 1986, 205, 146. 10. Withers, S. G.; Street, I. P.; Bird, P.; Dolphin, D. H. J. Amer. Chem. Soc. 1987, 109, 7530. 11. Adamson, J.; Foster, A. B.; Hall, L. D.; Johnson, R. N.; Hesse, R. M . Carbohydr. Res. 1970, 15, 351. 12. Hall, L. D.; Johnson, R. N.; Adamson, J . B.; Foster, A. B. Can. J. Chem. 1971, 49, 118. 13. Street, I. P.; Armstrong, C. R.; Withers, S. G . Biochemistry 1986, 25, 6021. 14. Withers, S. G.; Rupitz, K.; Street, I. P. J. Biol. Chem. 1988, 263, 7929. 15. Megregian, S. In ColorimetricDetermination of Non-Metals: Chemical Analysis; Boltz, D. F.; Howell, J . Α., Eds.; Wiley: New York, 1978; 8, 109. 16. Phillips, L.; Wray, V. J. Chem. Soc. (B) 1971, 1618. 17. Gerig, J. T . In Biological Magnetic Resonance; Berliner, L. T.; Reuben, J., Eds.; 1978, 1, 139. RECEIVED April 11, 1989
