Plant Cell Wall Polymers - American Chemical Society

"suicide" inactivators for glycosidases. /?-Glucosidases play an ... group. The timing of this "attack" of the carboxylate group is probably such that...
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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,

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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

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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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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

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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