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INTRODUCTION |
Two
-glucosidases of apparent molecular mass 62,000 have been
purified from extracts of germinated barley grain (1-3) and can be
classified in the family 1 group of glycosyl hydrolases and related
enzymes (4). The two enzymes have been designated isoenzymes
I and
II, and have isoelectric points of 8.9 and 9.0, respectively. Amino
acid sequence analyses reveal a single amino acid difference in the
first 50 NH2-terminal amino acid residues, and the complete
amino acid sequence of isoenzyme
II has been deduced from the
nucleotide sequence of a corresponding cDNA clone (2).
The function of the
-glucosidases in the germinated barley grain has
not been defined unequivocally, but will clearly be related to their
substrate specificities. It has been widely assumed that
-glucosidases prefer substrates of the type G-O-X, where G indicates the glucosyl residue and X can either be another
glycosyl residue, for which the linkage position is not crucial, or a
non-glycosyl aglycone group. As a result of the capacity of many
-glucosidases to hydrolyze glucosides with a range of glycosyl or
non-glycosyl aglycone groups, non-physiological substrates such as
4-nitrophenyl
-D-glucopyranoside
(4-NPG)1 have been
synthesized to measure activity in convenient spectrophotometric assays; the barley enzymes have also been assayed in this way. It has
further been assumed that the rate of hydrolysis of oligomeric substrates by
-glucosidases will remain approximately constant or
decrease with increasing degree of polymerization (DP) of the substrate
(5). However, the barley
-glucosidase isoenzyme
II hydrolyzes
(1,4)-
-oligoglucosides much more efficiently than it hydrolyzes the
aryl-
-glucoside 4-NPG (3). The increased hydrolytic rate with
oligosaccharides is a characteristic often observed with polysaccharide
exohydrolases (5), although the barley
-glucosidase is not able to
hydrolyze polymeric (1,4)-
-glucans (2, 3). These apparent anomalies
led Hrmova et al. (3) to suggest that the barley enzyme
could be classified either as a polysaccharide exohydrolase of the
(1,4)-
-glucan glucohydrolase group (EC 3.2.1.74) or as a
-glucosidase of the EC 3.2.1.21 class.
Because
-glucosidases are widely distributed in nature, a precise
understanding of substrate specificity and the mechanisms of substrate
binding and catalysis is essential for defining the functions and
tracing the evolution of this important group of enzymes. Here, subsite
binding energies of barley
-glucosidase isoenzyme
II have been
calculated from kinetic data during the hydrolysis of a series of
-oligoglucoside substrates. The analyses indicate that the enzyme
has at least six glucosyl-binding subsites. The catalytic amino acids
have been defined, together with their disposition in the
substrate-binding region. The substrate specificity, action pattern,
putative catalytic residues, and subsite mapping data can all be
reconciled with a three-dimensional model of the barley
-glucosidase, which is based on the x-ray crystal structure of an
homologous cyanogenic
-glucosidase from white clover (6).
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MATERIALS AND METHODS |
Chemicals--
The glucose diagnostic kit, 4-NPG, gentiobiose,
linamarin, bovine serum albumin (BSA), dithiothreitol, glycine ethyl
ester (GEE), and orcinol were purchased from Sigma. Conduritol B
epoxide was from ICN (Costa Mesa, CA), [14C]GEE was from
American Radiolabeled Chemicals (St. Louis, MO), L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin was from Worthington, trifluoroacetic acid was from Pierce, and Microcon microconcentrators were from Amicon (Beverly, MA). The Brownlee C18 guard column was obtained from Applied Biosystems (Foster
City, CA), a W-Porex C18 analytical column was from Phenomenex (Torrance, CA), Kieselgel 60 thin layer plates and sodium
2,2-dimethyl-2-silapentane-5-sulfonic acid were from Merck (Darmstadt,
Germany), chromatography paper no. 3MM Chr was from Whatman (Maidstone,
Kent, United Kingdom), and (1,4)-
-oligoglucosides (cellodextrins) of
DP 2-6 and (1,3)-
-oligoglucosides (laminaridextrins) of DP 2-7
were from Seikagaku Kogyo Co. (Tokyo, Japan).
Enzyme Purification--
Barley
-glucosidase isoenzyme
II
was purified from a homogenate of 8-day-old seedlings as described
previously (3). Its purity was assessed by SDS-PAGE, where a single
protein band was detected at high protein loadings. The purity was
further confirmed by NH2-terminal amino acid sequence
analysis; no secondary sequences were detected, and recoveries were
very close to theoretical values (data not shown).
Protein determination during the purification process, SDS-PAGE, and
amino acid sequence analyses were performed as described previously
(3).
Enzyme Assays and Kinetic Analyses--
Kinetic parameters on
(1,4)-
- and (1,3)-
-oligoglucosides were measured at 37 °C by
incubating 2-7 pmol of the purified
-glucosidase in 100 mM sodium acetate buffer, pH 5, containing 160 µg/ml BSA. Initial rates of hydrolysis were determined in triplicate at substrate concentrations ranging from 0.2 to 4 times the Km
value. Enzymic reactions were stopped by heating to 100 °C for 2 min, and released glucose was measured by the glucose oxidase method (7). Standard deviations, which ranged between 1% and 7%, were calculated (8), and kinetic data were processed by a proportional weighted fit, using a nonlinear regression analysis program based on
the Michaelis-Menten model equation (9). The initial enzyme concentration [E]0 was kept very much lower than the
initial substrate concentration [S]0, and care was taken
to measure initial reactions rates in all cases (10).
Initial hydrolysis rates of
-glucosidase isoenzyme
II with the
synthesized aryl-glycosides 4-NP-
-laminaribioside,
4-NP-
-cellobioside, and 4-NP-
-gentiobioside, and with 4-NPG as
substrates, were measured at 1 mM concentration. Substrate
solutions were prepared in 50 mM sodium acetate buffer, pH
5.0, using 
(300 nm) of 1.104
M
1 cm
1 to measure
concentrations, and were incubated with 4-5 pmol of the purified
-glucosidase at 37 °C. Enzyme activity was determined reductometrically by monitoring the increase in reducing sugars or
spectrophotometrically at 410 nm with 4-NPG (3). One unit of activity
is defined as the amount of enzyme required to release 1 µmol of
glucose from aryl-glycosides or to release 1 µmol of 4-nitrophenol
from 4-NPG per min. One unit corresponds to 16.67 nanokatals. Activity
on linamarin was measured as described by Barrett et al.
(6).
Calculation of Subsite Affinities--
Subsite affinities of
-glucosidase were calculated from Michaelis constants
(Km) and catalytic rate constants
(kcat) during the hydrolysis of
(1,4)-
-oligoglucosides of DP 2-6. In addition, the affinities of
subsites +2 and +3 for (1,3)-
-linked oligoglucosides were calculated
using Km and kcat values obtained with (1,3)-
-oligoglucosides of DP 2-4. The calculation procedures, using equations for the subsite analysis of exo-acting enzymes (11) but using subsite designations from
1 to +1, +2, etc.,
are summarized below.
Consider an active site consisting of x subsites, labeled
1, +1, ... to (x
1). The subsite affinity
Ai of the subsite labeled i (1 < i
(x
1)) can be calculated from
Equation 1.
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(Eq. 1)
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i=
1n(Ai)
and
i=
1n
1(Ai)
represent the sums of subsite affinities for subsites labeled from
1
to n and n
1, respectively. The parameters
(kcat/Km)n+1 and (kcat/Km)n are
determined for oligosaccharides of DP n + 1 and
n, respectively; R is the gas constant, and
T is the absolute temperature. The values
A
1 and kint are extrapolated from a plot of
exp(An/RT) versus
(1/kcat)n.
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(Eq. 2)
|
The vertical and horizontal intercepts of this equation are
exp(A
1/RT) and
1/kint. The parameter
kint is the intrinsic catalytic rate constant,
which is independent of DP. Finally, A+1 is
calculated from Equation 3.
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(Eq. 3)
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The validity of the subsite map was subsequently confirmed by
comparing the experimental and theoretical
kcat/Km values. The
theoretical values were calculated according to Equation 4.
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(Eq. 4)
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The equation uses the Ai and
kint parameters obtained for the
(1,4)-
-oligoglucosides (Table I). The comparison of the experimental
and theoretical kcat/Km
values gave a maximum deviation of 0.4%, which confirms the validity
of the model and the calculations of subsite affinities.
Glycosyl Transfer Reactions--
Freshly prepared 4-NPG (100 mM) in 20 mM sodium acetate buffer, pH 5.0, was
incubated with 2-3 pmol of the purified
-glucosidase for up to
72 h at 37 °C. The reaction was stopped by heating to 100 °C
for 2 min. Aliquots of the reaction mixture were separated by thin
layer chromatography on Kieselgel 60 thin layer plates and developed in
ethyl acetate/acetic acid/H2O (5:2:1, by volume). Reducing
sugars were detected using the orcinol reagent (12). Individual
products were scraped off the plates, eluted from the Kieselgel with
water, and their relative abundance determined spectrophotometrically
at 300 nm using 
of 1.104
M
1 cm
1.
For structural analysis of the transglycosylation products, the
reaction mixtures were scaled up 5 times and products were separated by
descending paper chromatography on Whatman no. 3MM paper in ethyl
acetate/acetic acid/H2O (3:2:1, by volume) at ambient temperature. The products were excised from the chromatogram, eluted
from the paper with water, concentrated under reduced pressure, and
analyzed using a Perkin-Elmer Sciex PAI 300 electrospray ionization triple quadrupole mass spectrometer (Perkin Elmer Sciex Instruments, Thornhill, Ontario, Canada) and by 13C NMR
spectroscopy.
13C NMR Spectroscopy--
13C NMR
spectra of oligosaccharides (3-8 µmol) were measured on a Varian
Gemini 300 multinuclear spectrometer using 5-mm external diameter
sample tubes at a probe temperature of 297 K. Transients (32) were
collected into 16,000 data points using a spectral width of 4.5 KHz and
a relaxation delay of 3 s with a 45° pulse width. No Gaussian
weight factor or line broadening was applied to the data before Fourier
transformation. Spectra were referenced and chemical shifts (ppm) were
given using sodium 2,2-dimethyl-2-silapentane-5-sulfonic acid as an
external standard.
Inactivation of
-Glucosidase by Conduritol B
Epoxide--
Inactivation of
-glucosidase isoenzyme
II was
monitored at 37 °C by incubating 57 pmol of purified enzyme in 100 mM sodium acetate buffer, pH 5.0, containing 160 µg/ml
BSA, with 0-10 mM conduritol B epoxide. To stop the
inactivation and to determine the residual activity at different times,
5-µl aliquots of the reaction mixture were diluted into 250 µl of
0.2% (w/v) 4-NPG in 100 mM sodium acetate buffer, pH 5.0, containing 160 µg/ml BSA. The residual enzyme activity was monitored
spectrophotometrically at 410 nm. First-order rate constants
(kapp) were determined from the semi-logarithmic
plots of residual activity as a function of time, using Equation 5.
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(Eq. 5)
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At and A0 are
enzyme activities at time t and 0, respectively. The
Ki and kmax constants were
determined and the order of the inactivation reaction was estimated
(13) to yield a inhibitor:enzyme stoichiometry of 0.7 (± 0.1) (data
not shown).
Identification of the Catalytic Nucleophile--
-Glucosidase
isoenzyme
II (43 µg) was inactivated at 37 °C by incubating the
purified enzyme in 100 mM sodium acetate buffer, pH 5.0, in
the presence of 24 mM conduritol B epoxide; this
corresponded to 4.4 times the Ki value. The
inactivated enzyme, which lost 99% of its activity over 5 h, was
concentrated, and excess inhibitor was removed in a microconcentrator
(exclusion limit 10,000).
Native and conduritol B epoxide-inactivated enzymes were denatured and
digested with trypsin, and the resulting tryptic peptides purified by
HPLC and sequenced essentially as described by Chen et al.
(14). The tryptic peptide-inhibitor conjugate (20 pmol), which was
unique to the conduritol B epoxide-inactivated enzyme, was treated with
20 µl of ammonium hydroxide at 50 °C for 20 min to remove
covalently bound conduritol. Excess ammonia was removed under vacuum,
and the peptide was again subjected to NH2-terminal amino
acid sequence analysis.
Identification of the Catalytic Acid--
Purified enzyme (57 pmol) was inactivated with 15 mM
1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) in the
presence of 125 mM [14C]GEE, the inactivated
enzyme was denatured and digested with trypsin, and the resultant
tryptic peptides were purified by HPLC for amino acid sequence analysis
(14). In addition, the molecular mass of the HPLC-purified
peptide-inhibitor conjugate was estimated on a Finnigan Lasermat
2000 matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Finnigan MAT, Hemel Hempstead, UK).
Hydrophobic Cluster Analysis--
Hydrophobic cluster analysis
(HCA) plots were obtained using standard computer software and
interpreted as described by Gaboriaud et al. (15). The amino
acid sequence of the barley
-glucosidase isoenzyme
II was taken
from Leah et al. (2), and the sequence of the cyanogenic
-glucosidase from white clover (Trifolium repens) was
obtained from the Brookhaven Protein Data Bank (entry 1cbg).
Protein Modeling--
Co-ordinates for a three-dimensional model
of the barley
-glucosidase were obtained using Swiss-Model, the
automated protein modeling service of the ExPasy Molecular Biology
Server (16-18). The barley enzyme sequence and co-ordinates of the
white clover
-glucosidase (6) (Brookhaven Data Bank entry 1cbg) were
supplied as inputs for the program. The three-dimensional model of
cellooctaose was constructed from the co-ordinates of cellobiose (19).
Fitting the cellooctaose model into the active site pocket of the
barley
-glucosidase model was performed on a Silicon Graphics Iris
Indigo Elan 4000 work station using the GRASP (20) and O (21) software programs. Co-ordinates of the Clostridium thermocellum
endoglucanase CelC were obtained from the Brookhaven Protein Data Bank
(entry 1cen; CelCE140Q-Glc6). Protein models
and stereoview diagrams were generated with the RasMol software program
(22).
 |
RESULTS |
Kinetic Analysis--
The relationship between the kinetic
parameters (Km, kcat, and
kcat/Km) and the DPs of
oligosaccharide substrates of the barley
-glucosidase isoenzyme
II are shown in Table I. For
cellodextrin substrates, Km decreases with
increasing chain length of the substrate, while
kcat values appear to be relatively independent
of DP, except in the case of cellotriose and to a lesser extent
cellotetraose, where kcat values are somewhat lower (Table I). Catalytic efficiency factors
kcat/Km increase steadily
with increasing DP of the (1,4)-
-oligoglucoside substrates, again
with the exception of cellotriose. Indeed, the kcat/Km value for
cellohexaose is 9-fold higher than the value for cellobiose.
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Table I
Kinetic parameters Km, kcat, and
kcat/Km of -glucosidase isoenzyme II during the
hydrolysis of cellooligosaccharides of DP 2-6 and
laminarioligosaccharides of DP 2-4
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In marked contrast to the hydrolysis of the cellodextrin series, the
kcat and
kcat/Km values for
laminaridextrins decreased by over 1000-fold and over 130-fold,
respectively, as the DP increased from 2 to 4 (Table I). The rate of
hydrolysis of laminaripentaose and longer (1,3)-
-oligoglucosides was
too low to allow the precise measurement of kinetic parameters. The enzyme had no activity on the cyanogenic substrate, linamarin.
Subsite Mapping--
Binding energies for the six
-glucosyl
binding subsites in the barley
-glucosidase isoenzyme
II during
hydrolysis of cellodextrins are compared in Fig.
1. Hrmova et al. (3)
reported that the enzyme catalyzes the hydrolytic removal of glucose
units from the non-reducing termini of oligosaccharide chains; this
demonstrated that the enzyme is an exohydrolase. It is clear,
therefore, that the catalytic amino acids are located between the
non-reducing terminal glucosyl-binding subsite and the penultimate
subsite; these are designated subsites
1 and +1, respectively (Fig.
1). Binding energies have been expressed in the past as positive or negative values (23-25), but we prefer to use positive values to indicate binding (10) (Fig. 1). The negative value observed at subsite
+2 indicates that there is a degree of repulsion between the enzyme and
the glucosyl residue at this subsite. The binding energies at subsites
+3, +4, and +5 are positive, but decrease in magnitude as the distance
from the catalytic site increases (Fig. 1).

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Fig. 1.
Subsite map of -glucosidase isoenzyme
II evaluated for the hydrolysis of (1,4)- -oligoglucosides.
The number of subsites and the position of the catalytic site were
determined according to Hiromi et al. (11). The
arrow indicates the position of catalytic amino acids.
Subsites are labeled from 1 to +5, where 1 represents the subsite
that binds the non-reducing terminal glucosyl residue of the substrate
and +5 is the subsite that binds the reducing end residue. The values
under the figure indicate the binding affinities for individual
subsites and the intrinsic catalytic rate constant
(kint).
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|
Although the barley
-glucosidase hydrolyzes laminaribiose at a
slightly faster rate than cellobiose, the rate decreases steeply as the
DP of the (1,3)-
-oligoglucoside substrates increase (Table I).
Calculation of the apparent binding energies of these laminaridextrins to subsites +2 and +3 yielded values of
4.2 and
8.4
kJ·mol
1, consistent with the low affinities and
kcat values of the enzyme for laminaritriose and
laminaritetraose (Table I).
The subsite mapping results (Fig. 1) offer an explanation for the
relatively low rate of hydrolysis of cellotriose (Table I). Based on
the binding energies of individual subsites, one might anticipate that
"non-productive" binding of cellotriose across subsites +1 to +3
would be almost as likely to occur on thermodynamic grounds as would
"productive" binding from subsites
1 to +2. Because the
non-productive binding does not span the catalytic site, hydrolysis
will not occur, but occupation of part of the substrate-binding region
would be expected to lower the catalytic rate, and hence the catalytic
efficiency (Table I). Non-productive binding of cellotetraose in
subsites +1 to +4 might also occur, but to a lesser extent.
Glycosyl Transfer Reactions--
When the barley
-glucosidase
was incubated with 100 mM 4-NPG, the major products were
glucose and 4-NP, as expected. However, significant amounts of other,
higher molecular weight 4-NP derivatives were visible on thin layer
chromatograms (data not shown). When the three most abundant of these
were eluted from the plates and analyzed, m/z values of
486.2 or 486.3 were obtained by electrospray mass spectrometry
(data not shown). These values correspond to disaccharide derivatives
of 4-NP. Chemical shifts obtained for 13C NMR spectroscopy
indicated that the disaccharide derivatives were
4-NP-
-laminaribioside, 4-NP-
-cellobioside, and
4-NP-
-gentiobioside (Table II).
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Table II
13C NMR chemical shifts of aryl-glycosides synthesized by
-glucosidase isoenzyme II during the glycosyl transfer reaction
of 100 mM 4-NPG
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The relative proportions of individual transglycosylation products are
compared in Table III. The
4-NP-
-laminaribioside was the most abundant, but significant levels
of 4-NP-
-cellobioside and 4-NP-
-gentiobioside were also detected.
This suggests that there is some flexibility in the binding of a
glucosyl residue to subsite +1, because the 4-NPG is able to move
sufficiently in the active site to present hydroxyls on C atoms 3, 4, or 6 to the bound glycone prior to transfer of that bound glucosyl residue to the 4-NPG (Scheme I). The
purified transglycosylation products were subsequently used as
substrates under standard conditions for hydrolysis. The specific
activities of hydrolysis were found to reflect, approximately at least,
their relative rates of synthesis under conditions that promote
glycosyl transfer reactions (Table III, column 3; cf. column
2).
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Table III
Summary of properties of glycosyl-transfer and other reaction products
produced by -glucosidase isoenzyme II during the glycosyl
transfer reaction of 100 mM 4-NPG
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Scheme I.
A hypothetical model of a glucosyl-enzyme
intermediate formed during the glucosyl-transfer reaction of
-glucosidase. A, hydrolysis of 4-NPG and the
formation of the glucosyl-enzyme intermediate. B, the
glucosyl-enzyme intermediate acts as a donor, while an acceptor is the
second 4-NPG molecule, giving rise preferentially to (1,3)- -,
(1,4)- -, and (1, 6)- -glycosidic linkages, as indicated by the
dashed arrows. The arrowhead positioned between
binding subsites 1 and +1 shows the position of catalytic amino acids
Glu181 and Glu391, and the open
arrow indicates the departing aglycone. The linkage between the
nucleophile and C-1 atom of the glucopyranosyl residue is marked by a
dashed line.
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Identification of Catalytic Amino Acid Residues--
Conduritol B
epoxide, or 1,2-anhydro-myo-inositol, has been used
extensively as a mechanism-based inhibitor to label the catalytic nucleophile of
-glucosidases (26). This compound inhibited the
barley
-glucosidase with a
kmax/Ki value of 0.2 s
1 M
1, and when tryptic
peptides of the inhibited enzyme were separated by HPLC, one new
peptide appeared (Fig. 2, peak
1). Amino acid sequence analyses of peaks 1 and 2 of the inhibited
and native enzymes showed that their sequences were identical, except
for a gap in the sequence of the inhibited enzyme at cycle 10. Following aminolysis, a glutamic acid residue appeared in this position during subsequent sequence analysis. It could be concluded that the
inhibitor had bound to amino acid residue Glu391 in the
native enzyme and that Glu391 is likely to be the catalytic
nucleophile. It should be noted that peak 3 derived from the native,
uninhibited enzyme was not present in digests of the inhibited enzyme
(Fig. 2). This peptide has a single glutamic acid residue which
corresponds to Glu212 in the native enzyme; the peak was
not detected after inhibition, and subsequent molecular modeling data
suggested that Glu212 does not play any role in
catalysis (data not shown).

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Fig. 2.
Comparative peptide mapping of tryptic
fragments of native and conduritol B epoxide-inactivated
-glucosidase isoenzyme II. Tryptic digests of native
(continuous line) and conduritol B epoxide-inactivated
(dotted line) enzymes were separated on a W-Porex C18
reverse-phase column and eluted from the column at a flow rate of 0.15 ml/min, using the gradient of 0.05% (v/v) trifluoroacetic acid
(solvent A) and 70% (v/v) CH3CN with 0.035% (v/v)
trifluoroacetic acid (solvent B). The eluent program at 0-5 min was
1% (v/v) solvent B and 99% (v/v) solvent A (isocratic); 5-75 min,
35% A and 65% B (linear); 75-85 min, 100% B (linear). The peptide
fractions 1-3, which are indicated by full and dashed
arrows and are unique to the digests of the native and inactivated
enzymes, respectively, were subjected to amino acid sequence
analysis.
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In attempts to identify the catalytic acid, the barley
-glucosidase
was labeled using the carbodiimide-nucleophile displacement procedure
(27). After labeling, enzymic activity was reduced by 93% and several
tryptic peptides with altered mobilities were isolated and sequenced.
Only one peptide had a blocked amino acid residue, and this
corresponded to Asp362 (data not shown). Mass spectrometric
analysis of the peak revealed an m/z of 1874, which is very
close to the value expected for the glycine ethyl ester adduct of that
particular tryptic peptide (data not shown).
Hydrophobic Cluster Analysis--
HCA of the barley
-glucosidase is compared with that of a cyanogenic
-glucosidase
from white clover (6) in Fig. 3;
comparisons were also made with other
-glucosidases (data not
shown). Similarities in the positions of hydrophobic clusters suggest
that
-helices and
-strands of the barley enzyme are likely to be
located in positions similar to those determined by x-ray
crystallography for the white clover
-glucosidase, as indicated in
Fig. 3.

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Fig. 3.
HCA plots of clover and barley
-glucosidases. Amino acid sequences can be read on the
near vertical rows, from the top left to the
bottom right. Hydrophobic clusters are boxed.
Vertical dashed lines in the white clover plot (A
and B) delineate the boundaries of the secondary structural
elements ( -strands and -helices) of the white clover
-glucosidase ( / )8 barrel. The catalytic acid and
the nucleophile are indicated by the arrows and are labeled
AH and B , respectively. The
arrowheads mark nine conserved residues of the catalytic
site (cf. Fig. 6). The Asp362 residue of the
barley enzyme, which was labeled by the EAC/GEE procedure, is not
conserved in other -glucosidases. Standard one-letter
codes for amino acids are used except the symbol is used for
Pro, for Gly, for Ser, and for Thr.
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Conserved acidic amino acid residues, in similar environments with
respect to clusters of hydrophobic amino acids, are detected at
Glu77, Glu181, Glu391, and
Glu445 for the barley
-glucosidase; corresponding
residues in the white clover
-glucosidase are Glu77,
Glu183, Glu397, and Glu453,
respectively. Amino acid residue Glu391 of the barley
enzyme was labeled with conduritol B epoxide (Fig. 2), but the
Asp362 that was labeled during the EAC/GEE modification is
not highly conserved in the sequences of
-glucosidases examined
here. Similarly, Glu residues in positions equivalent to
Glu445 of the barley enzyme were conserved only in about
50% of the 30
-glucosidase sequences extracted from the data bases
for alignment purposes in the present work, and Glu residues equivalent
to Glu77 are not highly conserved either (data not shown).
It might be concluded on this basis that the catalytic acid of the
barley
-glucosidase is likely to be the more highly conserved
Glu181, but there are also several highly conserved Asp
residues in the enzymes that might be involved (data not shown).
Protein Modeling--
The 3D-1D score (Fig.
4) gives a measure of the compatibility
of the predicted three-dimensional structure of the barley
-glucosidase, which was modeled on the white clover enzyme by the
ExPasy service, with the known primary sequence of the barley
-glucosidase isoenzyme
II. Here, the score varies between 0.2 and
1.0, with an average value of 0.65 (Fig. 4). The white clover
-glucosidase (6) shows 49% sequence identity with the barley enzyme
(Fig. 3). Scores below zero are obtained for incorrectly folded models,
although correctly folded models can still have average scores as low
as 0.4 (28). In a separate technique for evaluating the similarity in
protein folds (29), z-scores greater than 7 have been shown to indicate correctly identified folds. Using this procedure, a
z-score of 65 was obtained here for the prediction of barley
-glucosidase structure, again based on the white clover enzyme. Similarly, under PredictProtein a z-score of 7.3 was
obtained and may be compared with the value of 4.5, which is found to
give correct predictions in 88% of test cases (30, 31). Based on these
high reliability values, the model of the barley
-glucosidase was
also considered reliable and is shown in stereoview in Fig. 5.

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Fig. 4.
3D-1D plot. Structure-sequence
compatibility plot for the barley -glucosidase model built on the
x-ray crystal structure of the -glucosidase from white clover
(6).
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Fig. 5.
Stereo diagram of barley -glucosidase
space filling model illustrating catalytic residues and ten putative
residues involved in substrate binding. The blue, black,
purple, and green color indicate Tyr
(Tyr252, Tyr320, Tyr323,
Tyr338), Trp (Trp331, Trp342,
Trp363), Phe (Phe346, Phe454), and
His (His272) side chains, respectively. Asp362
is colored yellow. The likely catalytic residues
Glu181 and Glu391 are in red. The
drawing was generated with RasMol.
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DISCUSSION |
Kinetic analyses of
-glucosidase isoenzyme
II from barley
have shown that the enzyme rapidly hydrolyzes (1,4)-
-oligoglucosides and that the catalytic efficiency factor,
kcat/Km, increases as the DP
of the substrate increases (Table I). This preference for longer
(1,4)-
-oligoglucosides is not observed with
(1,3)-
-oligoglucosides. Although laminaribiose can be hydrolyzed
with relatively high kcat and
kcat/Km values, catalytic
rates and efficiencies decrease dramatically as the DP of this group of
substrates increases; the catalytic rate for laminaritetraose is
extremely low (Table I), and activity on longer
(1,3)-
-oligoglucosides in the series could not be measured. These
kinetic and substrate specificity data confirm the suggestion by Hrmova
et al. (3) that the barley enzyme could be classified as a
polysaccharide exohydrolase of the (1,4)-
-glucan glucohydrolase
group (EC 3.2.1.74), despite the fact that it does not hydrolyze
cellulosic substrates. The enzyme might be better described as a
cellodextrin glucohydrolase than as a (1,4)-
-glucan glucohydrolase.
Similar substrate specificities have been observed for the
celD gene product from Pseudomonas fluorescens
(32) and a
-glucosidase from the thermophilic bacterium Sulfolobus solfataricus (33). While noting the uncertainty
associated with the classification of the barley enzyme and related
enzymes from other sources, we have continued to refer to it as a
-glucosidase because it has sequence similarities to many other
enzymes that are classified as
-glucosidases in the protein and DNA
data bases. Once the native substrate of the enzyme has been identified
unequivocally, this nomenclature may be confirmed, or it may need to be
re-assessed.
The preference of the barley enzyme for (1,4)-
-oligoglucosides of
increasing chain length (Table I) suggested that it has an extended
substrate-binding region. This was examined and subsequently confirmed
by subsite mapping (Fig. 1). According to Hiromi and co-workers (11,
24), the substrate-binding region of polysaccharide hydrolases consists
of an array of tandemly arranged subsites, where each subsite binds a
single glycosyl residue of the polymer. Such an array of subsites has
now been demonstrated for endo- and exo-acting hydrolases and
glycosidases (10, 24, 34). If enzymes bind their polysaccharide
substrates via tandemly arranged subsites, it follows that kinetic
parameters will be dependent on the DP of the substrates. The
kcat/Km and
kcat values for oligosaccharides of increasing
chain length were therefore used to calculate binding affinities for
individual
-glucosyl-binding subsites on the barley
-glucosidase
(11).
At least six glucosyl-binding subsites were detected on barley
-glucosidase isoenzyme
II (Fig. 1). Binding energies are highest
at subsites
1 and +1 (Fig. 1), and although six subsites are shown in
Fig. 1, it is formally possible that additional subsites exist. The
insolubility of (1,4)-
-oligoglucosides of DP greater than 6 precluded the kinetic analyses required to test this possibility. The
significance of the negative energy term at subsite +2, which indicates
that glucosyl binding at this position is thermodynamically unfavorable, is not yet clear. However, similar negative binding energies have been observed at certain subsites in polysaccharide endohydrolases (10, 24).
Having confirmed the extended nature of the substrate binding region of
the barley
-glucosidase by subsite mapping, attention was shifted
toward the identification of the amino acid residues that mediate the
hydrolysis of the bound substrate. The catalytic residues are clearly
located between subsite
1, which binds the non-reducing terminal
glucosyl residue, and subsite +1, which binds the penultimate glucosyl
unit of the substrate, because it has previously been shown that the
enzyme releases glucose units from the non-reducing terminus of
oligomeric substrates (Fig. 1) (3). To identify the catalytic
nucleophile, the mechanism-based inhibitor conduritol B epoxide was
used (26, 35). This led to a dramatic reduction in activity and
sequence analysis of tryptic peptides from the inhibited enzyme showed
that the conduritol B epoxide had bound to amino acid residue
Glu391 (Fig. 2). To identify the catalytic acid,
carbodiimide-mediated labeling of the enzyme with GEE (27) was
attempted. The
-glucosidase was inhibited by the treatment and,
although some difficulty was experienced in tagging the enzyme, a
tryptic peptide was eventually isolated in which Asp362 was
labeled. Thus, the inhibitor studies indicated that Glu391
and Asp362 represented the catalytic nucleophile and the
catalytic acid, respectively, of barley
-glucosidase isoenzyme
II.
Given the difficulties that have been experienced in using any single
procedure to unequivocally identify amino acid residues that
participate in catalysis in polysaccharide hydrolases, hydrophobic cluster analyses and sequence alignments were subsequently used to
search for highly conserved acidic amino acids in
-glucosidases generally. Although the probable role of Glu391 in
catalysis was confirmed by these analyses, they suggested that
Glu181 was more likely than Asp362 to be the
catalytic acid (Fig. 3) and that the carbodiimide procedure might not
have correctly labeled the catalytic acid.
The revised identification of Glu181 as the catalytic acid
in the barley
-glucosidase was further investigated by molecular modeling. The primary structure of barley
-glucosidase isoenzyme
II (2, 3) was analyzed using the Swiss-Model molecular modeling
software program (16-18), in the expectation that the dispositions of
specific amino acid residues in the three-dimensional structure of the
enzyme would indicate whether or not they might participate in
catalysis. While acknowledging the limitations and constraints
associated with modeling, we obtained very high z-scores
when the barley
-glucosidase sequence was "fitted" to the
structure of the cyanogenic
-glucosidase from white clover (6) (Fig.
4) and, on the basis of these scores, the models shown in Figs. 5 and 7
were considered to be reliable. The most noteworthy feature of the
three-dimensional model of the barley
-glucosidase is the presence
of a deep slot, or pocket, in the surface of the enzyme. Close to the
bottom of the pocket are the probable catalytic amino acid residues
Glu181 and Glu391 (shown in red in
Fig. 5). The distance of 5.6 Å between their closest O atoms is widely
accepted as typical for retaining polysaccharide hydrolases (36, 37).
The Asp362 residue, which was tagged with GEE during
carbodiimide-mediated labeling, is located on the surface of the model,
near the entrance to the pocket, but some 20 Å from the catalytic
nucleophile (Fig. 6). This modeling
result confirmed that the carbodiimide procedure did not correctly
label the catalytic acid of the barley
-glucosidase. It also
indicated that other conserved Glu and Asp residues, such as
Glu77, which is also found in a conserved "hydrophobic"
environment (Fig. 3), are located too far from the catalytic
nucleophile to be direct participants in catalysis. Thus, the molecular
modeling programs proved to be a useful tool for the identification of amino acid residues that are likely to be involved in catalysis.

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Fig. 6.
Conserved amino acid residues in the active
site region of barley -glucosidase, C. thermocellum
endoglucanase, and barley (1,3;1,4)- -glucan
endohydrolase. Figure shows the conserved residues in the family 1 (A), family 5 (B), and family 17 (C).
The red residues are the catalytic residues and
non-catalytic Glu residues, the yellow residues are aromatic
residues, the green are His and Arg residues, and Asn is in
purple. The exceptions are His198 of family 5 (B) and Trp33 of family 17 (C), which are colored in
purple and green, respectively, indicating
structural replacements of the Asn and His residues at these positions.
The drawing was generated with RasMol, and one-letter amino acid
codes are used.
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As with most glycoside hydrolases, a number of highly conserved
residues are clustered near the catalytic amino acid residues. In Fig.
6, conserved residues or their functional equivalents in the active
sites of retaining glycoside hydrolases from family 1 (barley
-glucosidase), family 5 (endo-cellulase from C. thermocellum) (38), and family 17 (barley (1,3;1,4)-
-glucanase;
Ref. 39) are compared. These families are members of the so-called 4/7 superfamily of (
/
)8 glycoside hydrolases, all of
which have their putative catalytic acid located in an Asn-Glu pair
near the end of
-strand 4 and their catalytic nucleophile near the end of
-strand 7 (36, 40, 41). Of particular interest is the
presence of a conserved Glu residue about 8 Å from the catalytic nucleophile. This is Glu445 in the barley
-glucosidase
and Glu288 in the barley (1,3)- and
(1,3;1,4)-
-glucanases, but there is no equivalent Glu residue
conserved in the family 5 enzymes (Fig. 6). This residue
(Glu445 in the barley-
-glucosidase) is conserved in more
than 50% of data base entries for family 1 enzymes, although it is
absent in 6-phosphoglycosidases. It might be involved in substrate
binding very close to the catalytic site, in particular in binding the C-4(OH) of the non-reducing terminal glucosyl residue (6, 41).
The molecular model of the barley
-glucosidase could also be
reconciled with the substrate specificity and subsite mapping data. A
model of the (1,4)-
-oligoglucoside, cellooctaose, will fit into the
pocket (Fig. 7), but an equivalent
(1,3)-
-oligoglucoside molecule will not (data not shown). As far as
the subsite mapping data are concerned, use of the cellooctaose
molecule as a "molecular ruler" along the side of the pocket shows
that there is a potentially close association between the enzyme
surface and at least six glucosyl residues of the substrate (Fig. 7);
this is consistent with the presence of six glucosyl subsites in the
substrate-binding region of the enzyme (Fig. 1). The inside surface of
the putative substrate-binding pocket is also lined with several
aromatic amino acid residues (Fig. 5), which are likely to participate
in substrate binding through stacking interactions with the non-polar
surfaces of glucosyl residues (42).

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Fig. 7.
Molecular surface drawing of barley
-glucosidase with cellooctaose in its active site. A, a
model of cellooctaose is arranged with its non-reducing end at the
bottom of the substrate-binding pocket and lies along the longer side
(38 Å) of the funnel. The five glucosyl residues at the reducing end
can be seen protruding from the pocket; the three residues at the
non-reducing end within the pocket cannot be seen in the orientation
shown here. B, the approximate positions of the
glucosyl-binding subsites 1 to +5 along the bound cellooctaose.
Again, the non-reducing end of the oligosaccharide lies to the right
and is positioned between the catalytic residues Glu181 and
Glu391, which are marked in red. The molecular
surface of the protein is shown in blue, and the
oligosaccharide is represented as a wire model, where carbon atoms are
yellow and oxygen atoms are red. Where the
cellooctaose disappears from view in the pocket (subsites +2 to 1),
the molecule is shown in faded colors, to indicate that it
is being viewed "through" to the internal region of the enzyme.
Similarly, the catalytic residues at the bottom of the pocket are shown
in faded red, because they cannot be seen directly in the
orientation of the enzyme shown here. The drawing was generated with
GRASP; the bar indicates 5 µm.
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If the non-reducing terminus of the substrate is pushed to the bottom
of the pocket, where Glu445 and Trp446 are
located, the catalytic amino acids (Glu181 and
Glu391) are arranged close to the O atom of the first
glycosidic linkage. Thus, there is room for subsite
1 (Fig. 1)
between the catalytic residues and the bottom of the pocket. The pocket
broadens out above the catalytic residues, and this would explain not
only why a range of disaccharides can be hydrolyzed by the enzyme (2, 3), but the broadening above the catalytic residues would also allow
the limited movement of an incoming 4-NPG molecule that would be
necessary for the formation of (1,3)-, (1,4)-, and (1,6)-linked transglycosylation products (Table III). It is not clear why the non-reducing rather than the reducing end of the substrate is oriented
at the bottom of the pocket, but this is presumably related to
interactions of the enzyme with key hydroxyl groups of the non-reducing
terminal residue (43). Again, the Glu445 residue at the
very bottom of the pocket might be involved in this "orientation"
aspect of specificity, as might residues near the entrance to the
pocket.
There does not appear to be sufficient space for the non-reducing end
glucose unit to diffuse out from subsite
1 after hydrolysis, if the
substrate remains bound at subsite +1. Neither is there a "rear
exit" for the departure of released glucose units from the bottom of
the pocket, as there is for the release of the reaction product,
cellobiose, through the rear of the tunnel found in the family 6 exohydrolase, cellobiohydrolase II from Trichoderma reesei (37, 44, 45). In the case of the barley
-glucosidase, it seems
likely that the substrate is at least partially released from the
enzyme after each hydrolytic event, although the oligosaccharide product could remain inserted in the pocket for rapid re-binding prior
to the next hydrolytic event.
We conclude from these analyses that the three-dimensional model of the
barley
-glucosidase provides valuable clues as to the identity of
the catalytic amino acids, it can be reconciled with the presence of at
least six glucosyl-binding subsites, it offers an explanation not only
for the enzyme's ability to hydrolyze a broad range of small, dimeric
substrates but also for its preference for the relatively straight
(1,4)-
-oligoglucoside substrates, and it is consistent with the
observed transglycosylation reactions. The model also provides some
indications as to the natural substrates and hence to the possible
functions of the
-glucosidases in germinated barley grain. Barley
-glucosidases are synthesized in the starchy endosperm of developing
grain, but amounts of the enzyme do not increase substantially after
germination (1, 2). Leah et al. (2) have discussed in detail
some of the possible functions of
-glucosidases in germinated barley
grain. Perhaps the most likely function of the barley
-glucosidases
is in the complete hydrolysis of cell wall (1,3;1,4)-
-glucans in
germinated grain. These polysaccharides comprise about 70% of walls in
the starchy endosperm and constituent glucose residues account for as
much as 18% of total glucose in the grain (46). The
-glucosidases will not hydrolyze the parent polysaccharide (3), and this almost
certainly results from the irregularly spaced but relatively abundant
(approximately 30%) (1,3)-
-glucosyl linkages that are distributed
along the polysaccharide chain. The (1,3)-
-linkages introduce kinks
into the chain (47) that would prevent it fitting into the active site
pocket of the enzyme (Figs. 5 and 7). Initial hydrolysis of the
(1,3;1,4)-
-glucan therefore relies on endohydrolases, which release
as major products the trisaccharide
3-O-
-cellobiosyl-D-glucose (G4G3Gred) and the tetrasaccharide
3-O-
-cellotriosyl-D-glucose (G4G4G3Gred) (48). In addition, up to 10% of the
(1,3;1,4)-
-glucan is released by the endohydrolases as longer chain
(G4)nG4G3Gred oligosaccharides, where n
can be 2-10 (49). These oligosaccharides, which are essentially
(1,4)-
-oligoglucosides with a single (1,3)-linkage at their reducing
ends, have structures very similar to cellodextrins and would be
expected to fit into the active site pocket of the
-glucosidase
(Figs. 5 and 7). The
-glucosidases in germinated barley grain could
therefore play a major role in the reclamation of cell wall-bound
glucose as an energy source to support seedling growth.
We are grateful to Dr. Graham Jones, Dr. Neil
Shirley, Dr. Mark Duncan, Dr. Jose Varghese, Jelle Lahnstein, and Ann
Poljak for assistance with various aspects of the work.