Glycon specificity profiling of α-glucosidases using monodeoxy and mono-O-methyl derivatives of p-nitrophenyl α-D-glucopyranoside

Article abstract of DOI:10.1016/S0008-6215(02)00026-5

Hydrolysis of probe substrates, eight possible monodeoxy and mono-O-methyl analogs of p-nitrophenyl α-D-glucopyranoside (pNP α-D-Glc), modified at the C-2, C-3, C-4, and C-6 positions, was studied as part of investigations into the glycon specificities of seven α-glucosidases (EC 3.2.1.20) isolated from Saccharomyces cerevisiae, Bacillus stearothermophilus, honeybee (two enzymes), sugar beet, flint corn, and Aspergillus niger. The glucosidases from sugar beet, flint corn, and A. niger were found to hydrolyze the 2-deoxy analogs with substantially higher activities than against pNP α-D-Glc. Moreover, the flint corn and A. niger enzymes showed hydrolyzing activities, although low, for the 3-deoxy analog. The other four α-glucosidases did not exhibit any activities for either the 2- or the 3-deoxy analogs. None of the seven enzymes exhibited any activities toward the 4-deoxy, 6-deoxy, or any of the methoxy analogs. The hydrolysis results, with the deoxy substrate analogs, demonstrated that α-glucosidases having remarkably different glycon specificities exist in nature. Further insight into the hydrolysis of deoxyglycosides was obtained by determining the kinetic parameters (k_cat and K_m) for the reactions of sugar beet, flint corn, and A. niger enzymes.

Full text of DOI:10.1016/S0008-6215(02)00026-5

Carbohydrate Research 337 (2002) 629–634
www.elsevier.com/locate/carres
Note
Glycon specificity profiling of a-glucosidases using monodeoxy and
mono-O-methyl derivatives of p-nitrophenyl a- -glucopyranoside
D
Toshiyuki Nishio,a,* Wataru Hakamata,b Atsuo Kimura,c Seiya Chiba,c
Akira Takatsuki,b Ryu Kawachi,a Tadatake Okua
aLaboratory of Bio-organic Chemistry, Department of Biological Chemistry, College of Bioresource Sciences, Nihon Uni6ersity, 1866 Kameino,
Fujisawa-shi, Kanagawa 252-8510, Japan
bAnimal and Cellular Systems Laboratory, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198,
Japan
cDi6ision of Applied Bioscience, Graduate School of Agriculture, Hokkaido Uni6ersity, Nishi-9, Kita-9, Kita-ku, Sapporo, Hokkaido 060-8589,
Japan
Received 5 September 2001; received in revised form 18 January 2002; accepted 20 January 2002
Abstract
Hydrolysis of probe substrates, eight possible monodeoxy and mono-O-methyl analogs of p-nitrophenyl a-
D-glucopyranoside
(pNP a- -Glc), modified at the C-2, C-3, C-4, and C-6 positions, was studied as part of investigations into the glycon specificities
D
of seven a-glucosidases (EC 3.2.1.20) isolated from Saccharomyces cere6isiae, Bacillus stearothermophilus, honeybee (two
enzymes), sugar beet, flint corn, and Aspergillus niger. The glucosidases from sugar beet, flint corn, and A. niger were found to
hydrolyze the 2-deoxy analogs with substantially higher activities than against pNP a-D-Glc. Moreover, the flint corn and A. niger
enzymes showed hydrolyzing activities, although low, for the 3-deoxy analog. The other four a-glucosidases did not exhibit any
activities for either the 2- or the 3-deoxy analogs. None of the seven enzymes exhibited any activities toward the 4-deoxy, 6-deoxy,
or any of the methoxy analogs. The hydrolysis results, with the deoxy substrate analogs, demonstrated that a-glucosidases having
remarkably different glycon specificities exist in nature. Further insight into the hydrolysis of deoxyglycosides was obtained by
determining the kinetic parameters (kcat and Km) for the reactions of sugar beet, flint corn, and A. niger enzymes. © 2002 Elsevier
Science Ltd. All rights reserved.
Keywords: a-Glucosidase; Substrate specificity; Glycon specificity profiling; p-Nitrophenyl a-D-glucoside; Monodeoxy glucoside; Mono-O-methyl
glucoside
1. Introduction
glycosidic substrate is important in the formation of the
enzyme–substrate (ES) complex. Studies have reported
that various exo-type carbohydrate hydrolases, specifi-
cally a-glucosidase (EC 3.2.1.20) from barley malt,1
b-glucosidases (EC 3.2.1.21) from almond,2 molds As-
pergillus wetti,3 A. oryzae,4 A. niger,5 and bacteria
Agrobacterium faecalis,6 b-galactosidases (EC 3.2.1.23)
from the bacterium Escherichia coli7 and lamb small
intestine,8 and glucoamylase (EC 3.2.1.3) from A.
niger9–13 exhibit significant hydrolytic activities against
specific deoxy analogs of their corresponding glycosidic
substrates. We have also reported the hydrolytic activi-
ties of a-mannosidases (EC 3.2.1.24) from jack bean
and almond,14,15 a-glucosidase from rice,15,16 and a-
galactosidases (EC 3.2.1.22) from green coffee bean,
molds Mortierella 6inasea, and A. niger15,17 against the
Exo-glycosidases are a group of carbohydrate hydro-
lases that release monosaccharides from synthetic gly-
cosides and from the nonreducing ends of oligo-
saccharides and polysaccharides. The different types of
glycosidases are classified on the basis of specificities for
the glycon structure of their substrates, and their sub-
strate specificities are expressed in their relative activi-
ties against substrates having various aglycons. It has
been observed that hydrogen bonding between the ac-
tive site of the enzyme and the hydroxyl groups of the
* Corresponding author. Fax: +81-466-843951.
E-mail address: nishio@brs.nihon-u.ac.jp (T. Nishio).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00026-5

630
T. Nishio et al. / Carbohydrate Research 337 (2002) 629–634
four monodeoxy and four mono-O-methyl analogs of
the corresponding p-nitrophenyl (pNP) a- -glycopy-
strates are derivatives of pNP a-
(pNP a- -Glc, 1), as shown in Fig. 1. The four
monodeoxy analogs, modified at C-2, C-3, C-4, and C-6
are respectively, pNP 2-deoxy-a- -arabino-hexopyran-
oside (2), pNP 3-deoxy-a- -ribo-hexopyranoside (3),
pNP 4-deoxy-a- -xylo-hexopyranoside (4), and pNP
a- -quinovopyranoside (5). The four mono-O-methyl
derivatives modified at C-2, C-3, C-4, and C-6 are pNP
2-O-methyl-a- -glucopyranoside (6), pNP 3-O-methyl-
a- -glucopyranoside (7), pNP 4-O-methyl-a- -gluco-
-glucopyran-
D-glucopyranoside
D
D
ranosides. Significant hydrolytic activities were exhib-
ited by a-mannosidases toward the 6-deoxy substrate
D
D
analog, pNP a-
and a-galactosidases showed high activities toward
their 2-deoxy substrates, pNP 2-deoxy-a- -arabino-
hexopyranoside and pNP 2-deoxy-a- -lyxo-hexopyran-
D
-rhamnopyranoside, and a-glucosidase
D
D
D
D
D
oside, respectively. In contrast, the a-glycosidases
showed no activity toward the mono-O-methyl deriva-
tives, probably because of the hydrophobic character
and steric hindrance of the methoxy group. The obser-
vation that some deoxy substrates are hydrolyzed by
certain exo-type carbohydrate hydrolases suggests that
specific hydroxyl groups of sugar substrates play a
critical role in the hydrolysis reaction.
D
D
pyranoside (8), and pNP 6-O-methyl-a-
D
oside (9). The differences of the glycon specificities
among the enzymes are discussed.
2. Results and discussion
To gain further insight into the substrate-recognizing
mechanism in glycosidases, we have here investigated
the glycon specificities of various a-glucosidases (a-D-
The hydrolytic activities of the a-glucosidases from
the various sources against 1 and the monodeoxy (2, 3,
4, and 5) and mono-O-methyl (6, 7, 8, and 9) substrate
analogs were investigated under the conditions listed in
Table 1. Since different reaction conditions were used
for each enzyme, it is important to note that our
findings allow for only rough comparisons among the
activities of the enzymes and other a-glucosidases. The
investigations show that the four enzymes belonging to
family I (or family 13), from S. cere6isiae, B. stearother-
mophilus, and honeybee (honeybee I and III), did not
hydrolyze the deoxy analogs. In contrast, the three
enzymes, from sugar beet, flint corn, and A. niger,
showed hydrolytic activities against one or two deoxy
analogs. As observed for the rice enzyme,16 the sugar
beet a-glucosidase hydrolyzed only the 2-deoxy analog
2, however with substantially higher activity than
against 1. The enzymes from flint corn and A. niger
showed activities, not only toward 2 (substantially
higher than for 1 in both cases), but also toward the
3-deoxy analog 3 (although still lower than for 1 in
each case). None of the seven enzymes exhibited activi-
ties against the 4- and 6-deoxy analogs, 4 and 5. Since
every glycon hydroxyl group of 1 was shown to be
necessary for the hydrolytic actions of a-glucosidases
from S. cere6isiae, B. stearothermophilus, and honeybee,
while OH-2 does not appear to be essential for the
catalytic activities of the enzymes from rice, sugar beet,
flint corn, and A. niger, the deoxy substrate analogs
demonstrate that a-glucosidases with remarkably differ-
ent glycon specificities exist in nature. The enzymes
from S. cere6isiae, B. stearothermophilus, and honeybee
belong to family 13, whereas the enzymes of rice, sugar
beet, and A. niger belong to family 31.19,20 Based on its
amino acid sequence, we have classified flint corn a-glu-
cosidases as a member of family 31 (unpublished data).
The hydrolysis results for the deoxy substrate indicate
that a correlation exists between the glycon specificities
and the amino acid sequences of these eight a-
glucosidases.
glucoside glucohydrolase) against partially modified
substrates. Several a-glucosidases, widely distributed in
microorganisms, plants, insects, and animal tissues have
been purified and their amino acid sequences deter-
mined. Based on the conserved regions of the amino
acid sequences, these enzymes are divided into two
groups, the a-glucosidase families I and II.18 Addition-
ally, based on the amino acid sequence similarities,
these a-glucosidases are also classified as carbohydrate
hydrolases of family 13 or 31.19,20 The families I and II
a-glucosidases belong to the families 13 and 31 of
carbohydrate hydrolases. The enzyme described here,
rice a-glucosidase, which hydrolyzes the 2-deoxy glu-
coside, is classified in family II (or family 31). Herein,
we report the hydrolytic activities of four enzymes
classified in family I (or family 13): a-glucosidases from
yeast Saccharomyces cere6isiae, bacteria Bacillus
stearothermophilus, and honeybee (two enzymes), and
three enzymes classified in family II (or family 31),
namely the a-glucosidases from sugar beet, flint corn,
and A. niger. The partially modified hydrolytic sub-
Fig. 1. Structures of substrates used in this study.

T. Nishio et al. / Carbohydrate Research 337 (2002) 629–634
631
Table 1
Relative rates of hydrolysis on monodeoxy and mono-O-methyl derivatives of pNP a-
D
-glucopyranoside by each a-glucosidase
Enzyme source
Relative rate of hydrolysis (%) a
Compound
2
3
4
5
6
7
8
9
S. cere6isiae
B. stearothermophilus
Honeybee I
Honeybee II
Rice b
Sugar beet
Flint corn
A. niger
–
–
–
–
175
244
231
259
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3.7
11.9
Hydrolytic activity of each a-glucosidase was assayed under the following conditions: S. cere6isiae and B. stearothermophilus, in
50 mM sodium phosphate buffer (pH 7.0) at 37 °C; honeybee I, in 50 mM sodium acetate buffer (pH 5.0) at 35 °C; honeybee II,
in 50 mM sodium acetate buffer (pH 5.5) at 35 °C; sugar beet and flint corn, in 50 mM sodium acetate buffer containing 0.05%
Triton X-100 (pH 4.5) at 37 °C; A. niger, in 50 mM sodium acetate buffer (pH 4.0) at 37 °C. After the reaction was stopped by
addition of 0.3 M Na2CO3 solution, the amount of p-nitrophenol in each mixture was measured spectrophotometrically at 405
nm.
a Relative rate of hydrolysis was expressed by comparison with the amount of p-nitrophenol that was released from pNP
a- -glucoside (1) for 30 min by each a-glucosidase, which was taken as 100%.
D
b Data with rice a-glucosidase was taken from Ref. 16.
–, activity was too low to be measured.
Using the methoxy derivatives (6, 7, 8, and 9) as
substrates, none of the seven enzymes exhibited any
hydrolytic activity, similarly observed for rice a-glu-
cosidase.15 For these monomethylated glucosides, it
may be postulated that the hydrophobic character and
steric hindrance of the methoxy group prevented their
binding to the enzymes.
depressing formation of the carbonium–oxonium ion
of the reaction intermediate by its inductive effect.
Conversely, the lack of an OH-2 group in the deoxy
analog 2 destabilizes its glycosidic bond. These higher
kcat and kcat/Km values from the hydrolysis of 2 may
thus result from the instability of the glycosidic bond
in 2.
To further examine the details of the hydrolytic
activities of sugar beet, flint corn, and A. niger a-glu-
cosidases against the deoxy substrates, kinetic studies
on the hydrolysis of 1, 2, and 3 were carried out (Table
2). From the reaction velocities measured at different
substrate concentrations in each reaction mixture, the
kcat and Km values for each enzyme–substrate (ES)
combination were calculated using the Lineweaver–
Burk plot of 1/6 versus 1/S. The Km values obtained
using the enzymes from sugar beet, flint corn, and A.
niger against substrate 1 were 1.04, 0.88, and 0.59,
respectively, and against substrate 2 were 5.70, 7.38,
and 6.09, respectively. The Km values of the flint corn
and A. niger enzymes against substrate 3 were 9.98 and
10.2. In comparison to substrate 1, the higher Km
values shown by each enzyme toward substrates 2 or 3
are probably attributable to a partial loss of hydrogen
bonding, which is necessary in the formation of the ES
complex. Since the kcat or kcat/Km values of each en-
zyme for 2 were higher than those for 1, it can be
postulated that the C-2 hydroxyl group of 1 con-
tributes to the stability of its glycosidic bond, thereby
It has been reported that the OH-2 group of aryl
b-D-glucopyranoside substrates play an important role
in the catalytic reactions of some b-glucosidases.2–6
X-ray analysis and protein mutation studies of the
b-glucosidase of the bacterium Cellulomonas fimi indi-
cated that the glycon OH-2 contributes to transition-
state stabilization of
a covalent glycosyl–enzyme
intermediate.21,22 Moreover, a similar mechanism in-
volving OH-2 of the glycon has also been mentioned in
studies involving yeast a-glucosidase.23 In barley malt
a-glucosidase, every hydroxyl groups of the nonreduc-
ing end and OH-3 of the reducing end of maltose were
confirmed to be essential for the formation of hydro-
gen bonds between the enzyme and the substrate in the
transition-state complex.13 Our present studies indicate
that the glycon OH-2 of pNP a-D-glucopyranoside is
not involved in stabilizing the intermediate in the cases
of family II (family 31) a-glucosidases from sugar beet,
flint corn, and A. niger, or that from rice. Further
studies, including protein-engineering techniques,
should be carried out to elucidate details of glycon
specificities of a-glucosidases from different sources.

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T. Nishio et al. / Carbohydrate Research 337 (2002) 629–634

T. Nishio et al. / Carbohydrate Research 337 (2002) 629–634
633
Acknowledgements
3. Experimental
The authors express their sincere gratitude to Dr H.
Matsui of Hokkaido University for his support. This
work was supported in part by a Grant from the
Ministry of Education, Science, Sports, and Culture
(Japan) to promote advanced scientific research.
Enzymes and substrates.—Pure grade a-glucosidases
of S. cere6isiae and B. stearothermophilus were obtained
from Oriental Yeast Co. and Sigma–Aldrich Co., re-
spectively, and were further purified using ion-exchange
column chromatography (DEAE-Toyopearl 650 resin,
TOSO Co.) and gel-filtration column chromatography
(Toyopearl HW-55F resin, TOSO Co.). a-Glucosidases
from honeybee,24 sugar beet,25 flint corn,26 and A.
niger27 were purified as reported. Homogeneity of the
purified enzyme was confirmed using SDS- and native-
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sodium phosphate buffer (pH 7.0) at 37 °C; honeybee I,
in 50 mM NaOAc buffer (pH 5.0) at 35 °C; honeybee
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mM NaOAc buffer (pH 4.0) at 37 °C. After the addi-
tion of enzyme to substrate that was present at a
concentration of 1.0 mM in a final volume of 0.5 mL,
the mixture was incubated. After stopping the reaction
by the addition 0.3 M Na2CO3 solution (0.5 mL), the
amount of p-nitrophenol in the mixture was measured
spectrophotometrically at 405 nm. Kinetic studies of
the enzyme reaction were carried out at substrate con-
centrations between 0.5 and 2.5 mM, and the values of
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reaction curves and the molar concentrations of the
enzymes. Molecular weights of the enzymes are as
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