Substrate recognition by three family 13 yeast a-glucosidases
Evaluation of deoxygenated and conformationally biased isomaltosides
Torben P. Frandsen
1,
*, Monica M. Palcic
2
and Birte Svensson
1
1
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark;
2
Department of Chemistry,
University of Alberta, Edmonton, Canada
Important hydrogen bonding interactions between s ubstrate
OH-groups i n yeas t a-glucosidases a nd oligo-1,6-glucosidase
from glycoside hydrolase family 13 have been identi®ed by
measuring the rates of hydrolysis of methyl a-isomaltoside
and its seven monodeoxygenated analogs. The transition -
state stabilization energy, DDGà, contributed by the indi-
vidual OH-groups was calculated from the activities for
the parent and the deoxy analogs, respectively, according
to DDGà  ±RT ln[(V
max
/K
m
)
analog
/(V
max
/K
m

hydrolase family 31, respectively.
Keywords: protein-carbohydrate interaction; NMR; glyco-
sidase mechanism; substrate analogs; molecular recognition.
Strong intermolecular hydrogen bonds are very important
in speci®city of enzymes and o ther proteins that metabolize
or bind carbohydrates [1±6]. Substrate analogs such as
deoxygenated sugars, facilitate identi®cation of critical
contacts and enable quanti®cation of the energetics of the
protein±carbohydrate binding at the level of individual
interacting sugar OH-groups and functional atoms or
groups in the protein [4,7±11]. Alternatively, site-speci®c
mutants of a protein are useful in evaluation of speci®c
protein±carbohydrate i nteractions and further insight has
been gained by combining mutant enzymes and analogs
[7,9,10]. The binding energy contributed by substrate
OH-groups has been determined for only a few carbohy-
drate active enzymes. Of these, the starch hydrolase
glucoamylase from Aspe rgillus n iger has been the most
intensively examined [7,9±13].
Three-dimensional structures of protein±carbohydrate
complexes can guide and support protein engineering and
molecular r ecognition experiments. For family 13 glycoside
hydrolases, there are no c rystal structures for a-glucosidases;
however, t he structure o f free Bacillus oligo-1,6-glucosidase
has been solved [14]. Furthermore, only a few a-glucosidases
are produced by heterologous gene expression, which is a
prerequisite for structure±function relationship i nvestiga-
tions by site-directed mutagenesis [15±21]. While the yeast
genome is known and thus the primary structures of its
a-glucosidases, the sequenced strain of Saccharomyces

D
-glucan glucohydrolase, EC 3.2.1.3).
*Present address: Pantheco, Bùge Alle
Â
, DK 2970 Hùrsholm Denmark.
Dedication: this paper is de dicated to Prof. Joachim Thiem on the
occasion of his 60
th
birthday.
(Received 12 O ctober 2001, revised 26 November 2001, accepted 30
November 2001)
Eur. J. Biochem. 269, 728±734 (2002) Ó FEBS 2002
di- a nd oligo-saccharides and starch at comparable rates
[23,24]. The sequence classi®es a-glucosidases in glycoside
hydrolase families 13 and 31 [25±27]. Yeast a-glucosidases
and oligo-1,6-glucosidase belong to family 13 and are
of type I that prefers p-nitrophenyl-a-
D
-glucopyranoside
[28].
Glycoside hydrolase family 13 (or Ôthe a-amylase familyÕ)
currently comprises 28 speci®cities of amylolytic and related
enzymes. Several crystal structures of enzyme-inhibitor
complexes highlight active sites created by b ® a segments
in c atalytic (b/a)
8
barrel domains (reviewed in [29±31]).
Because no ligand complex is available of oligo-1,6-
glucosidase, the only structure-determined exo-acting
a-glucosidase [14], s ide-chains partic ipating in s ubstrate

retaining glycoside hydrolase family 31 [9,10,40,41].
MATERIALS AND METHODS
Enzymes and substrates
Oligo-1,6-glucosidase from baker's yeast (EC 3.2.1.10;
Lot no. 23H8080), and a-glucosidases from brewer's
(EC 3.2.1.20; Type VI; Lot no. 21F8 105) and b aker's
(EC 3.2.1.20; Type I; Lot no. 122H8000) yeast were
obtained from Sigma. After dissolution in 50 m
M
phosphate
pH 6.8 (a-glucosidases) or 50 m
M
sodium maleate pH 6.8
(oligo-1,6-glucosidase) followed by extensive dialysis at 4 °C
against these buffers, the different enzymes (oligo-1,6-
glucosidase, 30 UámL
)1
; brewer's yeast a-glucosidase,
200 UámL
)1
; baker's yeast a-glucosidase, 61 UámL
)1
)were
used without further puri®cation in the kinetic and stereo-
chemical studies. One unit i s de®ned as t he amount of
enzyme required to liberate 1 lmol of glucose from
p-nitrophenyl a-
D
-glucoside (Sigma) p er min at 30 °C. The
synthesized methyl a-isomaltoside, seven monodeoxy-

lysed essentially as described [10,40,41] with substrate
analogs at t he nonreducing e nd sugar ( reaction volume
400 lL) aliquots (100 lL) were transferred to quench buffer
containing 60 UámL
)1
glucose oxidase, 1 UámL
)1
peroxi-
dase, and 0.1 mgámL
)1
o-dianisidine, and the absorbances
were read at 450 nm after 4 h incubation at room
temperature, and quanti®ed using the relevant deoxygenated
D
-glucose as standard. The a-glucosidase catalyzed hydro-
lysis was initiated by a ddition of 0.1±91 U enzyme. The
limited amounts of deoxygenated analogs available allowed
only determination of second-order rate constants, V
max
/K
m
(s
)1
áU
)1
)  v
o
/E
o
S

log and b to parent substrate. For the two diastereoisomers,
V
max
and K
m
were determined by ®tting initial rates at eight
different substrate concen trations from 0.1 ´ K
m
to 4 ´ K
m
to the Michealis±Menten equation essentially as described
previously [40].
Reaction stereochemistry
Lyophilized enzymes w ere redissolved in 0.1
M
sodium
phosphate pH 6.8 in D
2
O and the stereochemistry of
isomaltose hydrolys is was determined by
1
HNMRat
310 K using a Bruker AMX-600 spectrometer operated at
600 MHz. After recording the substrate spectrum of
100 m
M
isomaltose (in 600 lL0.1
M
phosphate, pH 6.8,
in D

showed ninefold, ®vefold, and no reduction in V
max
/K
m
,
respective ly (Ta ble 1).
The DDGà calculated from the V
max
/K
m
values deter-
mined for a given analog and the parent substrate,
respectively, indicated the energy contributed to transition-
state stabilization by corresponding the OH-group. Because
DDGà for the four deoxy-analogs at t he nonreducing sugar
ring, that binds to the enzymes at subsite )1, was in the
range 16.1±24.0 kJámol
)1
for the three enzymes (Table 1),
the r emoval of one of the OH-groups from this ring
dramatically affected substrate hydrolysis. These
OH-groups can therefore be considered key polar groups
and m ost likely interact w ith c harged residues on t he
proteins [44] (Fig. 1 ). At the reducing end ring, however,
DDGà values of 4±6 kJámol
)1
for oligo-1,6-glucosidase
(Table 1) were obtained by replacement of the OH-2
and -3 groups, respectively, suggesting that these
OH-groups participate in neutral hydrogen bonds with the

+1 in type III a-glucosidases, exhibit substrate speci®city
variation among the a-glucosidases. The yeast a-glucosid-
ases as reported h ere only show protein±carbohydrate
hydrogen bonding involving subsite )1,andnosugar
OH-groups associated stabilization energy was critical for
accommodation at subsite +1. As shown in Table 2, these
a-glucosidases that do not require hydrogen bonding to the
Table 1. Speci®city constants and DDGà
a
(kJámol
)1
)fora-glucosidase catalyzed hydrolysis of methyl a-isomaltoside and a series of mono-deoxy-
genated analogs.
Oligo-1,6-glucosidase
b
a-glucosidase (brewer's yeast)
c
a-Glucosidase (baker's yeast)
d
V
max
/K
m
(s
)1
áU
)1
) DDGà V
max
/K

)6
5.8 3.7 ´ 10
)5
 4.9 ´ 10
)6
0.5 9.7 ´ 10
)5
 5.8 ´ 10
)6
2.3
3-Deoxy-methyl-a-isomaltoside 2.9 ´ 10
)5
 0.6 ´ 10
)6
4.1 6.0 ´ 10
)5
 1.4 ´ 10
)5
)0.8 1.3 ´ 10
)4
 1.3 ´ 10
)5
15
4-Deoxy-methyl-a-isomaltoside 1.4 ´ 10
)4
 1.1 ´ 10
)5
± 5.7 ´ 10
)5
 0.8 ´ 10

)9
23.9
4¢-Deoxy-methyl-a-isomaltoside 1.0 ´ 10
)7
 1.1 ´ 10
)8
19.1 1.5 ´ 10
)8
 4.4 ´ 10
)10
21.2 3.2 ´ 10
)8
 2.8 ´ 10
)9
23.5
6¢-Deoxy-methyl-a-isomaltoside 3.2 ´ 10
)7
 2.1 ´ 10
)8
16.1 7.9 ´ 10
)8
 4.7 ´ 10
)9
16.7 1.4 ´ 10
)7
 1.5 ´ 10
)8
19.6
a
DDGà  )RT ln[(V

, only f or oligo-1,6-glucosidase. Invariant glycoside hydrolase family
13 side chain candidates of interaction with the four nonreducing
substrate ring O H-groups are described in detail in a recent review [30].
730 T. P. Frandsen et al. (Eur. J. Biochem. 269) Ó FEBS 2002
substrate aglycon also have much higher activity for
p-nitrophenyl-a-
D
-glucopyranoside, which lacks hydrogen
bonding groups in the aglycon, than for isomaltose. Due t o
effects on b oth k
cat
and K
m
yeast a-glucosidase thus has
4500-fold lower k
cat
/K
m
for i somaltose than for p-nitrophe-
nyl-a-
D
-glucopyranoside, p-nitrophenol being also a better
leaving group than g lucose. Structural e lements of t he
nonsugar aglycon, however, were not explored. It is
conceivable, however, that such speci®city e xists and could
be investigated using a series of synthetic substrates. In
contrast, the activity of oligo-1,6-glucosidase signi®cantly
depends on aglycon interactions at subsite +1 via neutral
hydrogen bonds with glucose OH-2 and -3 (Table 1). That
such protein interactions with sugar OH-groups are impor-

hydrolysis by glucoamylase, which depended on enzyme±
substrate transition-state interactions with OH-4 and -3 of
an energy of 16.5 and 8.6 kJámol
)1
, respectively (Table 2;
[7]). Glucoamylase thus has only sixfold lower k
cat
/K
m
for
isomaltose than for p-nitrophenyl-a-
D
-glucopyranoside
(Table 2). The substrate speci®city d ifferences and varia-
tions in aglycon-protein contacts with the two a-glucosid-
ases, the oligo-1,6-glucosidase, and glucoamylase emphasize
that these enzymes display different geometry for the binding
interactions with polar groups of substrates at subsite +1.
This will be investigated further in a study of the diastereo-
isomer speci®city of isomaltoside h ydrolysis (see below).
Catalytic mechanism
One f eature of the d isposition o f substrate relative to
enzyme during the various steps of the catalytic events
directly relates to the mechanism of catalysis being funda-
mentally different for retaining and in verting enzymes [38].
The stereochemistry of isomaltose hydrolysis by yeast oligo-
1,6-glucosidase and a-glucosidases was con®rmed to involve
retention of the substrate anomeric con®guration in the
product. This is illustrated for baker's yeast a-glucosidase
which shows

b
Isomaltose 5.2 34.5 0.15
p-Nitrophenyl-a-
D
-glucopyranoside 135 0.2 677
OH-2 0.5
OH-3 )0.8
OH-4 )0.7
Oligo-1,6-glucosidase
a
Isomaltose 33.3 6.9 4.8
p-Nitrophenyl-a-
D
-glucopyranoside 129 1.3 988
OH-2 5.8
OH-3 4.1
OH-4 ±
Glucoamylase
c
Isomaltose 0.41 19.8 0.021
p-Nitrophenyl-a-
D
-glucopyranoside 0.50 3.7 0.135
OH-2 1.1
OH-3 8.6
OH-4 16.5
a
Data from Table 1;
b
[28];

analyses are not feasible due to the limited amounts of
analogs available; we therefore cannot determine the role of
a key polar group in the glycosylation or the deglycosylation
steps in the mechanism (Fig. 3). However, one can conclude
that the discrimination of the diastereoisomer, as this is
associated with the V
max
and not the K
m
, does not happen in
the initial reversible part of substrate complex formation,
but in subsequent steps o f the catalytic m echanism [40].
Recognition of diastereoisomeric isomaltoside
derivatives
Isomaltose is ¯exible due to rotation around the C5±C6
bond. It is possible to block this conformational ¯exibility
by alkylation of C6 (Fig. 4). Previously, methyl 6-R-and
methyl 6-S-methyl-a-isomaltoside were used to determine
the preferred rotational conformer for glycoamylase [40].
Hydrolysis catalyzed by baker's yeast a-glucosidase (this
enzyme was chosen as it has the highest activity of the two
a-glucosidases; see Table 1) was similarly examined using
methyl 6-R-ethyl- and methyl 6-S-ethyl-a-isomaltoside as
the pair of conformationally biased substrate analogs
(Table 3). While methyl 6-S-ethyl-a-isomaltoside was
hydrolyzed with twofold lower V
max
,butthesameK
m
as

ethyl-a-isomaltoside (A) and methyl 6 -S-ethyl-
a-isomaltoside (B).
Table 3. Kinetic parameters for the hydrolysis of conformationally biased isomaltosides.
Substrate V
max
(m
M
á s
)1
áU
)1
) K
m
(m
M
) V
max
/K
m
(s
)1
áU
)1
)
a-Glucosidase from baker's yeast
a
Isomaltose 2.8 ´ 10
)3
9.8 2.8 ´ 10
)4

Methyl 6-R-methyl-a-isomaltoside 0.68 0.71 0.96
a
At 30 °C, using 50 m
M
phosphate, pH 6.8.
b
[40].
732 T. P. Frandsen et al. (Eur. J. Biochem. 269) Ó FEBS 2002
spatial distribution of the groups that play an important
role in the enzyme recognition. This ®nding stresses the
fundamentally different active site architecture that exists
for the inverting glucoamylase and the retaining a-gluco-
sidases. Glucoamylase, in c ontrast to a-glucosidase, applies
a single d isplacement mechanism and belongs to a different
fold family, glycoside hydrolase 15. The speci®c activities
and substrate af®nities are similar for these retaining and
inverting enzymes, all of which have reasonable capacity i n
the g lucose release from the no nreducing end of disac-
charides and small substrates. However, the a-glucosidase
showed large variation in rate of hydrolysis between the
methyl 6-S-and6-R-ethyl a-isomaltosides, with small
differences in af®nity for the two distereoisomers, whereas
the discrimination b y glucoamylase was associated with the
K
m
[40] and not with the r ate of hydrolysis (Table 3).
CONCLUSION
The enzyme preparations used in the present analysis are
considered valuable representatives of two categories of
yeast a-glucosidases. The study strongly demonstrates the

substrate ring at subsite + 1 in the oligo-1,6-glucosidase as
well as for controlling the exo-action at th e level of the
nonreducing end ring at subsite )1ofallthreea-glucosid-
ases included in the present comparison. The ®ndings on
substrate key polar groups and preferred isomaltoside
diasteroisomers, however, will be valuable in future mod-
eling of s ubstrate complexes of the a-glucosidases a nd
related enzymes if the structures become available. The data
may thus guide protein engineering studies that address the
a-1,4 and a-1,6 bond speci®city of these closely related
a-glycosidases.
ACKNOWLEDGEMENTS
Bent O. Petersen is gratefully acknowledged for performing the NMR
spectroscopy experiments and Ulrike Spohr and Raymond U. Le mieux
are t hanked for t he synthetic substrate analogs. Th is work was
supported in part b y funding from the Natural Sciences and
Engineering Research Council of Canada (to M. M. P).
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