573
Glycosidase mechanisms
Carl S Rye and Stephen G Withers*
Insights into glycosidase mechanisms have come from X-ray
crystallographic studies on complexes with substrate analogs
and inhibitors, representing all the intermediate species along
the reaction coordinate. Site-directed mutagenesis continues
to play a significant role in understanding mechanisms, but is
also proving important in generating glycosidases of modified
mechanism or specificity.
Addresses
University of British Columbia, 2036 Main Mall, Vancouver, British
Columbia V6T 1Z1, Canada
*e-mail: withers@chem.ubc.ca
Current Opinion in Chemical Biology 2000, 4:573580
1367-5931/00/$ see front matter
2000 Elsevier Science Ltd. All rights reserved.
Introduction
It is only within the past two years that the true catalytic
prowess of glycosidases has been fully exposed through
the studies of Wolfenden on the spontaneous hydrolysis of
polysaccharides [1]. Rate enhancements of up to
1017-fold are typical, placing glycosidases amongst the
most proficient of enzymes. During the past two to three
years, the major mechanistic advances have been in the
structural analysis of enzymeligand complexes, in the
elucidation and investigation of variations on standard
mechanisms, in the modification of mechanisms and
specificity through site-directed mutagenesis, and in the
generation of new inhibitors. After a brief introduction,
this review focuses primarily on recent developments in
these areas. More detailed accounts are found in a number
of recent reviews [210].
The sequence-based classification of glycosidases continues
to be an extremely valuable tool in our understanding of
structure/function relationships, and the number of families
continues to grow, being approximately 80 at last count.
This information is currently available on a well-designed
Web site (http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html)
[11,12] that has recently been extended to include glycosyl
transferases, polysaccharide lyases, and carbohydrate
esterases and binding modules. Three-dimensional struc-
tures are now available for representatives of approximately
30 of these 80 families. New family structures published
since the last comprehensive list [7] include family 3 [13],
47 [14], 48 [15] and 77 [16].
There are two possible stereochemical outcomes for the
hydrolysis of a glycosidic bond: inversion or retention of
anomeric configuration. Both mechanisms (Figure 1)
involve oxacarbenium-ion-like transition states and a pair
of carboxylic acids at the active site. In inverting glycosi-
dases, these two residues are located approximately 10
(+/ 2 ) apart on average [5,17] and the reaction occurs via
a single-displacement mechanism wherein one carboxylic
acid acts as a general base and the other as a general acid
(Figure 1a). In retaining enzymes, the two carboxylic acid
residues are approximately 5.5 apart [5,17] and the reaction
proceeds via a double displacement mechanism (Figure 1b).
Mechanistic insights from structural studies
A particularly enlightening report is that by Davies et al.
[18] showing X-ray crystal structures of the four stable
states along the enzymatic reaction coordinate of a retain-
ing family 5 -glycosidase. These were the native,
substrate-bound, covalent intermediate, and product com-
plexes of Cel5A from Bacillus agaradhaerens. As had been
seen for the bound substrate (Michaelis complex) in other
cases [19,20], the sugar in the 1 subsite is distorted into a
1S skew-boat conformation. This can be interpreted
3
either as consistent with the dictates of stereoelectronic
theory, in which the lone pair of the endocyclic oxygen is
antiperiplanar to the departing glycosidic bond, or as sim-
ply minimizing steric hindrance suffered by the attacking
nucleophile from interactions with H3 or H5. By contrast,
the 1 subsite sugar in the catalytically competent
2-deoxy-2-fluoro--cellobiosyl-enzyme intermediate sits
in a relaxed 4C1 chair conformation, similar to other
trapped intermediates reported [21,22]. A water molecule
is located in an ideal position for nucleophilic attack on the
anomeric center above the pyranoside ring and is hydro-
gen-bonded to the acid/base catalytic residue. Finally, in
the cellotriose product complex, the diffuse electron den-
sity for the sugar in the 1 subsite is consistent with weak
binding of the product [18].
Initially surprising was the observation of a distorted 2,5B
(boat) conformation for the covalent 2-deoxy-2-fluoro--
xylobiosyl-enzyme intermediate on the retaining family 11
-1,4-xylanase from Bacillus circulans [23], and subse-
quently on the xylanase from B. agaradhaerens ([24];
Figure 2a). Further consideration led to the realization that
this conformation allows atoms C5, O5, C1, and C2 of the
sugar to achieve the coplanarity required at the oxacarbe-
nium-ion-like transition states that bracket this
intermediate. Thus, family 11 xylanases appear to effect
hydrolysis through a boat transition state (Figure 2b),
rather than the half chair that appears to be used by glu-
cosidases and cellulases. In those cases, a boat
conformation would place the bulky C5 hydroxymethyl
substituent in a disfavoured axial position. This hypothesis
explains the absolute xylan specificity of family 11 and the
looser cellulose/xylan specificity of family 10 enzymes,
which are believed to go through a half chair transition
state. Another interesting feature of this structure is an
interaction between the sugar endocyclic oxygen and a
conserved tyrosine hydroxyl that may serve to stabilize the
transition state or perhaps destabilize the ground state
574 Mechanisms

Figure 1

(a)
O O
O O O O
H H
O O
O + O
O R 10.5 HOR
R
H
O O
H OH

H H

O O O O O OH

(b)

O O O O
H H
O O
+
O
O R
R
5.5

O O O
O
O O
H
O
O
H
O O

HO O O O
H
O O
O +
OH
H

O
O O O

Current Opinion in Chemical Biology

General glycosidase mechanisms for (a) an inverting -glycosidase and (b) a retaining -glycosidase proceeding through an intermediate with
a 4C1 conformation.

structures. Interactions of a similar type with the ring and electrostatically at the transition state as the sugar ring
oxygen have been seen with nucleoside hydrolases [25]. flattens and develops positive charge.

Hydrogen bonding interactions with the sugar 2-hydroxyl
have been shown previously to be important for catalysis,
contributing 810 kcal mol1 to transition state stabiliza-
tion [7,8,26]. Direct observation of such interactions within
the glycosyl-enzyme intermediate has been achieved by
trapping a true cellobiosyl-enzyme intermediate on a dou-
ble mutant (His205Asn/Glu127Ala) exoglycanase, Cex,
from Cellulomonas fimi [21]. A short (2.4 0.2 ) hydrogen
bond was observed between the 2-hydroxyl and the car-
bonyl oxygen of the Glu233 nucleophile. This observation
led to the suggestion that this interaction (more than that
with the conserved Asn126, which mutagenesis studies
have shown contributes only 35 kcal mol1 to transition
state stabilization [27,28]) is responsible for most of the sta-
bilization afforded at the 2-position. Presumably, the
interaction with Glu233 is optimized both geometrically
The first structural characterization of an intermediate
trapped on an -glycosidase or -glycosyl transferase was
reported recently [29]. Because the 2-fluorosugar strategy,
in which a glycosyl-enzyme intermediate is trapped
through treatment with an acitvated 2-deoxy-2-fluorogly-
coside (the fluorine serving to destabilise the
oxacarbenium ion transition states) does not work with
-glycosidases, a different mode of intermediate trapping
was required. An ideal enzyme for this turned out to be the
cyclodextrin glycosyltransferase (CGTase) from Bacillus
sp. This enzyme is actually a member of glycohydrolase
family 13, a group dominated by -amylases and -glu-
cosidases, but carries out an intramolecular
transglycosylation reaction, carving off oligosaccharides of
six, seven or eight glucose residues from starch and releas-
ing them as cyclodextrins. Glycosyl fluorides are known
 Glycosidase mechanisms Rye and Withers 575

Figure 2

Conformations of enzyme-bound species.

(a) 2,5B (boat) conformation of the (a) Enzyme
2-deoxy-2-fluoro--xylobiosyl-enzyme
intermediate formed with a family 11 H
O O
xylanase. (b) The corresponding proposed H
O
boat transition state for hydrolysis of the HO O
HO O
glycosylenzyme intermediate. HO HO Enzyme
OH
O F
O

Enzyme

(b)

Enzyme

O O
H
H
H
O
+ +
RO Od
HO H
O
H
O

O

Enzyme
Current Opinion in Chemical Biology

substrates for this enzyme, undergoing transglycosylation
to produce linear and cyclic oligosaccharide products. By
removing the nucleophilic 4-hydroxyl group of the glyco-
syl fluoride, it proved possible to accumulate a
4-deoxy-glycosyl enzyme intermediate whose lifetime was
prolonged sufficiently to allow crystallographic studies by
the use of a mutant of the acid/base catalyst (Glu257Gln).
The -linked glucose residue in the 1 subsite adopts an
undistorted 4C1 conformation, suggesting that stereo-elec-
tronic assistance afforded by an antiperiplanar lone pair
arrangement is not an important factor in the breakdown of
this intermediate. A small amount of distortion towards a
half chair conformation was, however, seen for the sugar in
the 1 subsite in a complex of maltononaose with an
inactive double mutant (Glu257Gln/Asp229Asn), as well
as in a -cyclodextrin complex [30].
Mechanistic abnormalities
A variation of the general mechanism for retaining enzymes
has been demonstrated for the N-acetyl--hexosaminidases
belonging to families 18 and 20. In these enzymes, the sub-
strate N-acetyl group takes the place of the enzyme
nucleophile, attacking at the anomeric center and forming
an oxazolinium intermediate, as shown in Figure 3. Such a
mechanism is well precedented for acid-catalyzed hydroly-
sis of N-acetylglucosaminides [31] and has been considered
for enzymes previously [7]. Consistent with this, crystal
structures of enzymes from these families show no suitably

Figure 3

General mechanism for retaining

-N-acetylhexosaminidases, operating via an
oxazolinium ion intermediate.
O O O O O O
H H
O O
O O
O R H OH
HN
HN O HN + O O

CH3 CH3 CH3

Current Opinion in Chemical Biology
576 Mechanisms
Figure 4
OH
OH
HO O
HO O O
HO OH
HO
OH O
O OH
HO OH
OH 1,5-Anhydro-D-fructose (enol form)
OR
OH
O
HO
HO
O
1,5-Anhydro-D-fructose (keto form)
Current Opinion in Chemical Biology
positioned enzyme nucleophile [20,32]. Rather, a carboxy-
late group appears to be positioned suitably to stabilize the
oxazolinium intermediate. Strong support for acetamido
participation has come from structural studies of a chitinase
complex with allosamidin, a natural product that appears to
mimic the oxazoline [33]. Other support comes from the
crystal structure of the family 20 chitobiase from Serratia
marcescens in complex with its substrate, chitobiose [20].
The GlcNAc residue in the 1 subsite is bound in a dis-
torted skew-boat conformation with the carbonyl oxygen of
the N-acetamido group lying in an appropriate position for
attack at the anomeric center.
Support for the mechanism has also come from solution
studies. Firstly, GlcNAc oligosaccharides missing an
N-acetyl group at the normal site of cleavage were shown
to act as inhibitors for, rather than substrates of, the family
20 chitobiase from S. marcescens [34], pointing to the impor-
tance of the N-acetyl moiety. Secondly, the stable
thiazoline analogue of the oxazoline was shown to be an
excellent competitive inhibitor (Ki = 280 nM) of jack bean
N-acetyl--hexosaminidase [35]. Finally, and perhaps most
convincingly, Kobayashi et al. [36] succeeded in synthesiz-
ing the GlcNAc oxazoline and showed it to function as a
donor in the enzymatic synthesis of N,N-diacetylchitobiose
using a Bacillus sp. enzyme.
Not all retaining N-acetyl--hexosaminidases follow the
anchimeric assistance mode of glycoside bond cleavage.
Recent work by Vocadlo et al. [37] has shown that a gly-
cosyl-enzyme intermediate is formed on the family 3
-N-acetylglucosaminidase (Exo II) from Vibrio furnisii.
Reaction of the enzyme with the mechanism-based
reagent 2-acetamido-2-deoxy-5-fluoro--L-idopyranosyl
fluoride, which allows the accumulation of the inter-
mediate, followed by peptic digestion, liquid
chromatography/tandem mass spectrometry (LC/MS MS)
analysis, and sequence alignment identified Asp242 as
the catalytic nucleophile.
Reaction catalyzed by the -1,4-glucan lyases.
OH
+ HO
HO
OR
A particularly intriguing recent discovery was the exis-
tence of a class of enzymes that are related in sequence to
the retaining family 31 -glycosidases. These new
enzymes also cleave glucose residues from the non-reduc-
ing end of starch, but utilize an elimination reaction, as
opposed to hydrolysis, releasing 1,5-anhydro-D-fructose
([38]; Figure 4). Such enzymes can therefore be classi-
fied as -glucan lyases, but they are quite distinct from
the other polysaccharide lyases, which remove a proton
from C5, adjacent to a C6 carboxyl moiety, releasing a
4,5-unsaturated sugar. In the family 31 case, an un-acti-
vated proton at C2 is removed and a double bond forms
between C1 and C2; the reaction presumably follows an
E1-like mechanism involving an oxacarbenium ion
species. This would explain the structural and mechanis-
tic similarities to -glycosidases, and is consistent with
inhibition of -glucan lyases by known glycosidase
inhibitors such as 1-deoxynojirimycin and acarbose.
Another glycosidase family that stands out mechanistically is
family 4. The enzymes in this family are unique among gly-
cosidases as they require both NAD+ and a divalent metal
ion (such as Mn2+, Ni2+, Co2+, or Fe2+) for catalysis. Family 4
is also the only family that contains both - and -glycoside-
specific enzymes. NAD+ does not appear to be consumed
during the reaction; thus its role in catalysis is unclear, as is
the actual catalytic mechanism. Results of site-directed
mutagenesis studies, however, coupled with comparative
sequence alignments, have identified putative catalytic car-
boxylic acid residues [39,40]. X-ray crystal structures of both
- and -glycoside-specific enzymes of family 4 are needed
to gain greater understanding of the catalytic mechanism(s)
and either confirm or refute the assignment of the active-site
catalytic carboxylic acid residues [41].
Moulding mechanisms and shaping specificity
through mutagenesis
Techniques for identification of key active-site residues
based upon detailed kinetic analysis of mutants modified at

Figure 5
Glycosynthase mechanism with the
Glu358Ser mutant of Agrobacterium sp.
-glucosidase.
O O
F
those positions (see [4]) have been applied to several gly-
cosidases. Thus, the acid/base residue in galactanase A
from Pseudomonas fluorescens was identified through studies
with substrates of different leaving group ability [42],
whereas the identity of the active site nucleophile of the
-glycosidase from Sulfolobus solfataricus was confirmed as
Glu387 through studies on mutants with activated sub-
strates in the presence of sodium azide; a glycosyl-azide
product of inverted anomeric stereochemistry was formed
[43]. Further studies on the conversion of T4 lysozyme
from an inverting to a retaining enzyme mechanism with
transglycosylation activity have been reported using a
Thr26His mutant [44]. It was speculated that a mechanism
involving a covalent glycosyl-imidazole is involved; however,
a neighbouring group participation mechanism involving an
oxazolinium ion intermediate would seem more likely.
Mutagenesis studies on the general base catalyst of the
inverting glucoamylase from Aspergillus awamori have pro-
vided an enzyme with activities up to 160% of that of the
wild-type [45]. The serendipitous oxidation of the cys-
teine of the Asp400Cys mutant to cysteinesulfinic acid
(Cys400 SO2H) yielded this higher activity enzyme in
which the two catalytic residues are an estimated 1.2 fur-
ther apart than in the wild-type enzyme. Measurements of
pH dependence revealed that the pKa of the catalytic base
Cys400SO2H is 0.5 pH units lower than that of wild-type
[46]. In addition, the mutant was able to catalyze the self-
condensation of -D-glucopyranosyl fluoride and the
subsequent hydrolysis of the product to -glucose accord-
ing to a Hehre resynthesis/hydrolysis mechanism, under
conditions for which this was not detected for the
wild-type enzyme.
Specificity appears to be harder to change than mechanism
through site-directed mutagenesis. However, a recent
paper shows a double mutant of the xylanase 10A from
Pseudomonas cellulosa with significantly improved specificity
for the hydrolysis of glucose-derived substrates compared
with that of their xylose equivalents [47]. Success in
changing specificity has also been achieved in the absence
Glycosidase mechanisms Rye and Withers 577
O
O HO O
H
O O
O O
OR
OR
F H
H
O O
Current Opinion in Chemical Biology
of structural information by using directed molecular evo-
lution, involving multiple rounds of mutagenesis,
functional screening, and amplification. An excellent
example of this has been provided by Zhang et al. [48]
using PCR-based DNA shuffling. After seven rounds of
shuffling, a 1000-fold increase in the specificity for a chro-
mogenic fucoside substrate over that for a galactoside was
obtained using the Escherichia coli (lacZ) -galactosidase.
Although the bulk of the specificity change was effected
through a destruction of galactosidase activity, there was a
significant (10- to 20-fold) increase in fucosidase activity.
Useful changes in glycosidase specificity through directed
molecular evolution should be expected in the next few
years given the relative ease of screening these enzymes
and the large number of families known.
A useful application of mutagenesis has been the devel-
opment of a new method of oligosaccharide synthesis.
This involved using a mutant of a -glucosidase from
Agrobacterium sp., wherein the nucleophilic carboxyl had
been modified to alanine, in conjunction with activated
glycosyl fluorides of the opposite anomeric configuration
to that of the normal substrate [49]. The mutated gly-
cosidase (glycosynthase) catalyzes the synthesis of
glycosidic bonds, but is incapable of their hydrolysis, thus
allowing yields of over 90% to be achieved (Figure 5). A
recent development is a new glycosynthase containing
serine in place of the nucleophile that results in 24-fold
faster synthesis and thus improved product yields and
reduced reaction times [50]. A likely role for the serine
hydroxyl group is in hydrogen bonding with the departing
anomeric fluoride. Endoglycosidases have also been
developed as glycosynthases, allowing oligosaccharyl
fluorides to act as glycosyl donors [51,52].
An alternative to the use of -glycosyl fluoride donors in
the glycosynthase methodology has been demonstrated by
Trincone and co-workers [43,53] with the -glucosidase
from the thermophilic S. solfataricus. Using a nucleophile
mutant (Glu387Gly) of this enzyme in combination with
sodium formate and an activated -glucoside, a transient
578 Mechanisms
Figure 6
(a) (b)
OH
OH
O
HO O O
HO O HO
R HO
Enzyme
OH
OH
H O
O O O O
Enzyme Enzyme
Current Opinion in Chemical Biology
1-O-formyl glucose intermediate is formed in the enzymes
active site that then acts as the glycosyl donor, affording
-linked products that include branched structures.
Insights from inhibitors
The great catalytic proficiency of glycosidases alluded to ear-
lier demands very high transition state affinities, on the order
of 1022 M [1]. This suggests that very tight binding transi-
tion state analogue inhibitors of glycosidases are possible,
with potential as therapeutic agents. Interest in glycosidase
inhibitors as therapeutics has been heightened in recent
years with the successful marketing of the -amylase
inhibitor acarbose for control of blood glucose levels and of
the sialidase inhibitor Relenza as a treatment for influenza.
Rationalization of the inhibitory potencies of a series of
bicyclic derivatives of nojirimycin led Heightman and
Vasella [9] to the realization that glycosidases can be classi-
fied into two groups on the basis of whether the acid
catalyst protonates the glycosidic oxygen from a direction
anti or syn to the C1O5 bond (Figure 6). Such lateral pro-
tonation, essentially in the ring plane, has been seen in
X-ray crystal structures of several enzyme/substrate com-
plexes [9] as well as in the complex of Cel5A from
B. agaradhaerens with a cellobiose-derived imidazole [54].
Supporting kinetic evidence has come from studies of
Trichoderma cellulases [55] and from comparisons of family
10 and 11 xylanases with a series of xylobiose-derived aza
sugars, which are nanomolar inhibitors of the family 10
enzymes (anti-protonators), but only millimolar inhibitors
of those from family 11 (syn-protonators) [56]. However,
probable differences in sugar transition state conformation
between the two enzymes cloud the analysis.
Transition state analysis through the measurement of mul-
tiple kinetic isotope effects has resulted in the design,
synthesis, and testing of several valuable inhibitors. Most
notable have been the iminoribitol inhibitors of nucleoside
hydrolases and ribosyltransferases developed by the
Schramm and Furneaux groups [25,57,58,59,60].
Through a combination of crystallographic studies on
enzyme/inhibitor complexes and analysis of aglycone speci-
ficity, considerable success has been achieved in the design
Protonation trajectories of the glucosidic
Enzyme bond: (a) anti and (b) syn.
O O
H
O
R
of isozyme-specific inhibitors that bind up to 105-fold
tighter than the substrate. Fascinating insights into the
roles of individual hydrogen bonds have come from the
measurement, and in one case [60] the assignment, of low
field 1H NMR signals associated with such interactions.
Conclusions
Structural studies of complexes of various sorts have pro-
vided the major mechanistic insights into this class of
enzymes in the past 23 years and can be expected to con-
tinue to contribute, particularly in the studies of
enzyme/inhibitor complexes. While site-directed mutage-
nesis will continue to be a major tool in investigating roles
of individual side chains, directed evolution will play an
important role in the generation of enzymes with new
specificities and possibly even new mechanisms.
Acknowledgements
The authors would like to thank the Natural Sciences and Engineering
Research Council of Canada and the Protein Engineering Network of
Centres of Excellence of Canada for financial support.
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have been highlighted as:
of special interest
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29. Uitdehaag JCM, Mosi R, Kalk KH, van der Veen BA, Dijkhuizen L,
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This paper (see also [30]) provides the first structural characterization of an
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57. Miles RW, Tyler PC, Evans GB, Furneaux RH, Parkin DW,
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58. Shi WX, Li CM, Tyler PC, Furneaux RH, Cahill SM, Girvin ME,
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59. Shi WX, Li CM, Tyler PC, Furneaux RH, Grubmeyer C, Schramm VL,
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X-ray crystal structure relating to [60].
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This paper outlines the design of inhibitors based on the proposed transition
state for the reaction. 1H NMR analysis of the enzymeinhibitor complex
shows a number of downfield signals that can be related to strong hydrogen
bonds formed in the complex.