From the
Oligo-1,3-
The 1,3-
These possibilities suggest that a
detailed knowledge of substrate specificities of the
1,3-
The three-dimensional structure of barley
1,3-
To
confirm that tritium was introduced only into the reducing terminal
glucosyl residues, the oligosaccharides were reduced with NaBH
For the
quantitative kinetic analyses of hydrolysis of the tritiated
laminaridextrins, lower substrate concentrations were used, and
relatively higher concentrations of enzymes were required to allow
detection of the hydrolysis of shorter laminaridextrins. Even higher
concentrations of isoenzyme GIII were required because of its much
lower specific activity. Substrate and enzyme concentrations used for
the three 1,3-
In the calculation of the
kinetic parameter, k
On-line formulae not verified for accuracy where [S]
On-line formulae not verified for accuracy where G
The
bond-cleavage frequency of a specific glycosidic linkage is represented
by the fraction of particular labeled products released by the enzyme.
By plotting
On-line formulae not verified for accuracy at a given time, where G
On-line formulae not verified for accuracy and for these calculations we used bond-cleavage frequencies
To calculate the binding affinities of
subsites adjacent to the catalytic site, the total affinity of the two
subsites on either side of the catalytic site was calculated from the K
In quantitative
kinetic analyses, more sensitive assays based on radioactive substrates
were used, substrate concentrations were maintained in the low
micromolar range, enzyme concentrations were in the nanomolar range,
and reactions were monitored from 90 s onwards. These conditions were
chosen to ensure that initial reaction rates were measured throughout (). Rates were shown to be linear for at least 15 min (Fig. 1). For oligosaccharides shorter than DP 7, where
hydrolysis rates were relatively slow, enzyme concentrations were
increased to ``force'' the rate of hydrolysis to measurable
levels (). The kinetic parameters k
Subsite mapping of polysaccharide endohydrolases allows the
determination of the number of individual glycosyl binding subsites
involved in enzyme-substrate association, definition of the position of
the catalytic site in relation to the glycosyl binding subsites, and
the calculation of binding energies at each subsite. Two types of
experimental input are required, namely quantitative bond-cleavage
frequencies as a function of chain length and the kinetic parameter k
The kinetic analyses allow the determination of the parameter k
Against this theoretical background we have used subsite mapping to
describe enzyme-substrate binding in three 1,3-
The negative
binding energies observed for the glucosyl residues on either side of
the catalytic site (Fig. 5) are commonly observed in
polysaccharide endohydrolases and may cause substrate distortion in the
region of the glycosidic linkage that is hydrolyzed (Phillips, 1966;
Thoma et al., 1971). The binding energies shown in Fig. 5suggest that an incoming 1,3-
In summary, we have used subsite mapping procedures
to describe certain aspects of substrate-binding in three
1,3-
We are grateful to Professor M. A. Long for tritiation
of laminaridextrins, to Professor B. A. Stone for his encouragement and
advice, and to Dr. P. M. Colman for his continuing support. We are
particularly indebted to Dr. Jose Varghese and Professor Peter B.
Hfor their assistance and enthusiastic discussions.
Note Added in Proof-B. Henrissat, G. Davies, and co-workers
(personal communication) have used hydrophobic cluster analysis to
predict that Glu
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-glucosides with degrees of polymerization of
2-9 were labeled at their reducing terminal residues by catalytic
tritiation. These substrates were used in detailed kinetic and
thermodynamic analyses to examine substrate binding in
1,3-
-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes
GI, GII, and GIII from young seedlings of barley (Hordeum
vulgare). Bond-cleavage frequencies, together with the kinetic
parameter k
/K
,
have been calculated as a function of substrate chain length to define
the number of subsites that accommodate individual
-glucosyl
residues and to estimate binding energies at each subsite. Each
isoenzyme has eight
-glucosyl-binding subsites. The catalytic
amino acids are located between the third and fourth subsite from the
nonreducing terminus of the substrate. Negative binding energies in
subsites adjacent to the hydrolyzed glycosidic linkage suggest that
some substrate distortion may occur in this region during binding and
that the resultant strain induced in the substrate might facilitate
hydrolytic cleavage. If the 1,3-
-glucanases exert their function
as pathogenesis-related proteins by hydrolyzing the branched or
substituted 1,3;1,6-
-glucans of fungal walls, it is clear that
relatively extended regions of the cell wall polysaccharide must fit
into the substrate-binding cleft of the enzyme.
-glucan glucanohydrolases (EC 3.2.1.39), or
1,3-
-glucanases, are distributed widely in higher plants. Their
functions in normal plant growth and development appear to be
restricted to specialized roles in the removal of wound or dormancy
callose, in pollen tube growth, in microsporogenesis, or in senescence
(Stone and Clarke, 1993). However, as members of the
``pathogenesis-related'' group of proteins that are expressed
in higher plants in response to fungal, bacterial, or viral attack
(Boller, 1987), they are believed to be critically important in the
plant's defenses against potentially pathogenic microorganisms.
Considerable research attention has been focused on this aspect of
1,3-
-glucanase function (Stone and Clarke, 1993; Dixon and Lamb,
1990; Kauffmann et al., 1987). The enzymes are thought to
participate in the defense process by hydrolyzing the 1,3- and
1,3;1,6-
-glucans that are important constituents of cell walls in
many fungi, including plant pathogens (Wessels, 1993). In vitro studies show that 1,3-
-glucanases, especially in the presence
of chitinases, can cause extensive breakdown of walls in fungal hyphae
and that this can lead to cell lysis (Mauch et al., 1988;
Skriver et al., 1991). Alternatively, the 1,3-
-glucanases
may only partially hydrolyze fungal wall
-glucans, releasing
oligosaccharides that could elicit a variety of other plant defense
responses (Dixon and Lamb, 1990).
-glucanases may be important in defining their mode of action
in plant-pathogen interactions. If 1,3-
-glucanases were to
participate in a general defense strategy against a broad spectrum of
fungal pathogens, it might be expected that they would readily
hydrolyze branched or substituted 1,3;1,6-
-glucans of the type
found in fungal walls. However, in comparative work on the
specificities of barley 1,3-
-glucanase isoenzymes GI, GII, and
GIII, Hrmova and Fincher(1993) found that rates of hydrolysis of
branched or substituted 1,3;1,6-
-glucans of fungal origin were
relatively low.
-glucanase isoenzyme GII has now been determined by x-ray
crystallography, which revealed that the two catalytic amino acids,
Glu
and Glu
(Chen et al., 1993a),
are located within a deep substrate-binding groove which extends across
the surface of the enzyme (Varghese et al., 1994). It has also
been suggested that the three-dimensional conformations and the
positions of catalytic amino acids are likely to be conserved in all
higher plant 1,3-
-glucanases (Varghese et al., 1994). As
part of an ongoing objective to fully describe substrate binding,
specificity, catalytic mechanisms, and functions of
1,3-
-glucanases in precise molecular terms, we have now examined
the enzyme using subsite mapping procedures, in which the
substrate-binding site of a polysaccharide hydrolase can be considered
as an array of tandemly arranged subsites, where each subsite binds a
single glycosyl residue of the polysaccharide substrate (Hiromi, 1970;
Thoma et al., 1970; Hiromi et al., 1973; Thoma and
Allen, 1976; Suganuma et al., 1978; Allen, 1980). The number
of binding subsites in three barley 1,3-
-glucanases, their
disposition in relation to the catalytic site, and binding energies
associated with each subsite have been defined.
Enzyme Isolation and Assay
Barley
1,3--glucanase isoenzymes GI, GII, and GIII were purified from
extracts of 14-day-old seedlings by fractional precipitation with
ammonium sulfate, ion exchange chromatography, chromatofocusing, and
size exclusion chromatography as described previously (Hrmova and
Fincher, 1993). Enzyme purity and identity were routinely checked by
SDS-polyacrylamide gel electrophoresis and NH
-terminal
amino acid sequencing. 1,3-
-Glucanase activity was determined
colorimetrically during enzyme purification by measuring glucose
equivalents released (Nelson, 1944; Somogyi, 1952) from a 0.2% (w/v)
solution of laminarin from Laminaria digitata (Sigma) in 50
mM sodium acetate buffer, pH 4.8, at 37 °C. One unit of
activity is defined as the amount of enzyme required to release 1
µmol of glucose equivalents
min
and
corresponds to 16.67 nanokatals. The specific activities of the
purified 1,3-
-glucanase isoenzymes GI, GII, and GIII were 192,
249, and 64 units/mg of protein, respectively.
Oligosaccharide Substrates
Laminaridextrins of
degree of polymerization (DP)(
)2-7 were
purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Laminaridextrins of
DP 8-11 were purified from a partial hydrolysate generated by
treatment of 3 g of the 1,3-
-glucan, pachyman, from Poria
cocos (generously provided by Professor B. A. Stone) with 100 ml
of 90% (w/w) formic acid at 100 °C for 4 h. The laminaridextrins of
DP 8-11 were purified by repeated chromatography on a 1.5
120-cm column of Fractogel TSK HW-40 (Merck, Darmstadt, Germany)
equilibrated in water at a flow rate of 8.1 cm
h
at 20-22 °C. Eluted carbohydrate was detected using the
orcinol-H
SO
method (Bruckner, 1955; Nazarova
and Elyakova, 1982). The identities and purities of the
oligosaccharides were also checked by their mobility on Kieselgel 60
thin layer chromatography plates developed in ethyl acetate/acetic
acid/H
O (2:2:1 or 3:2:1 by volume) and detected with the
orcinol reagent (Hrmova and Fincher, 1993).
Tritiation of Oligosaccharides
Laminaridextrins
were labeled at their reducing terminal C-1 atom by catalytic
tritiation with tritium gas in the presence of palladium oxide coated
on barium sulfate (Evans et al., 1974) and purified by size
exclusion chromatography on Fractogel TSK HW-40 as described above.
Tritium in column fractions was measured by liquid scintillation
counting at 30% efficiency, and the identities of tritiated
oligo-1,3--glucosides were checked by thin layer chromatography.
Developed plates were dried, sprayed with EN
HANCE tritium
enhancer (DuPont NEN), and exposed to Hyperfilm-
H or
Hyperfilm-
max film (Amersham International, Amersham, United
Kingdom) for 4-7 days. For the quantitation of radioactivity, the
thin layer chromatography plates were cut into 0.5-cm segments and
radioactivity measured by liquid scintillation counting using
Ultima-Gold scintillation mixture (Packard Instruments B. V.,
Groningen, The Netherlands). Specific radioactivities ranged from 3 to
17 TBq
mol
, which corresponded to 80-465
Ci
mol
. Radioactive impurities, which probably
originated by degradation of oligosaccharides during the tritiation
procedure (Evans et al., 1974), accounted for between 2 and 9%
of laminaridextrins of DP 2-8 and less than 15% of
oligosaccharides of DP 9-11; these contaminants were separated
from the oligosaccharides during thin layer chromatography.
(Hahn et al., 1992) and hydrolyzed with 2 M trifluoroacetic acid for 1 h at 120 °C. Neutralized
hydrolysates were separated by thin layer chromatography in
2-propanol/toluene/ethyl acetate/water (50:10:25:12.5 by volume), which
clearly resolved glucose and sorbitol, and plates were sprayed with
orcinol. Radioactivity was associated only with sorbitol, which was
consistent with specific tritiation of reducing terminal glucosyl
residues (data not shown).
Kinetic Analyses
To compare the rates of
hydrolysis of unlabeled laminaridextrins, 1 mM substrate in 50
mM sodium acetate buffer, pH 4.8, was incubated with a final
concentration of isoenzyme GII of 21.5 nM at 37 °C for up
to 90 min. Aliquots were removed for the reductometric measurement of
reducing sugars with the neocuproine reagent (Dygert et al.,
1965) and the extent of hydrolysis expressed as percent.
-glucanase isoenzymes are shown in .
Aliquots were removed at time intervals from 1.5 to 360 min, enzyme
action was terminated by heating at 100 °C for 5 min, and reaction
products separated by thin layer chromatography. After autoradiography,
plates were cut into 0.5-cm segments for quantitation of radioactivity
associated with each oligosaccharide.
/K
, the
hydrolysis can be regarded as a first-order reaction with a first-order
rate constant (k) which is equal to k
/K
(Matsui et
al., 1991). Because the initial enzyme concentration
[E]
[S]
K
, k
/K
can be
determined from the integrated equation,
and [S] represent
substrate concentrations at zero time and after enzyme addition,
respectively. By plotting the function,
represents the radioactivity
of a substrate of DP n in the aliquot taken for analysis and G
is the radioactivity of an
oligosaccharide of DPj, k
/K
can be determined from the slope of the curve (Suganuma et al., 1978; Matsui et al., 1991).
is the
radioactivity of an oligosaccharide of DP j, G
is radioactivity measured in a product
of DP i and n is the DP of the oligosaccharide
substrate, the slope of the curve represents the bond-cleavage
frequency (Allen and Thoma, 1976).
Thermodynamic Analysis
Subsite affinities (A) were calculated by analysis of
kinetic data for substrates of DP n and (n -
1), which are hydrolyzed to products of DP i and (i - 1), respectively (Suganuma et al., 1978). Because
the ratio of the various substrate-enzyme complexes reflects the
substrate affinity at each binding subsite j, the sum of
affinities
A
= B
,
where B
is the molecular binding affinity (Hiromi et al.,
1973). Thus,
0.013. Standard deviations were calculated according to Samuels(1991)
by taking into account data from pairs of positional isomers of
enzyme-substrate complexes.
of laminarinonaose (DP 9), because the
DP of the substrate must be greater than the total number of subsites
(Hiromi et al., 1973; Shibaoka et al., 1974; Suganuma et al., 1978; Biely et al., 1981). The K
of laminarinonaose (DP 9) was
determined by incubating 0.2-1.5 mM 1-
H-labeled oligosaccharide of specific activity 8.5
TBq
mol
with 9.3, 13.5, and 59.7 nM final concentrations of isoenzymes GI, GII, and GIII,
respectively, in 10 mM sodium acetate buffer, pH 4.8, at 37
°C. Hydrolysis was monitored as described above for other labeled
laminaridextrins. Data were processed with a nonlinear regression
analysis program based on Michaelis-Menten kinetics (Perella, 1988).
Relative Rates of Hydrolysis of
Laminaridextrins
In the initial analyses the rates of hydrolysis
of laminaridextrins were compared at 1 mM substrate
concentration and with 21.5 nM enzyme. Under these conditions,
no hydrolysis of laminaridextrins of DP 2-7 could be detected
after 60 min with the relatively insensitive reductometric assay.
However, laminarioctaose was hydrolyzed and the rate of hydrolysis of
oligosaccharides of DP 8-11 increased with DP (data not shown).
Although these results suggested that oligosaccharides of DP greater
than 8 were required before hydrolysis occurred, it must be emphasized
that substrate concentrations were relatively high and that an extended
period of hydrolysis was used. It is therefore likely that secondary
hydrolysis might have occurred, whereby products of the primary
hydrolytic cleavage were further hydrolyzed; this effect would be more
pronounced as the DP of the substrate increased.
/K
of the three
isoenzymes during hydrolysis of the series of laminaridextrins are
presented in . First-order plots of the time course of
hydrolysis of the laminaridextrins by isoenzyme GIII are shown in Fig. 1.
Figure 1:
First-order plots of the time course of
hydrolysis of 1-H-labeled laminaridextrins of DP 3-6 (L3-L6) by 1,3-
-glucanase isoenzyme GIII. The
conditions of hydrolysis are specified in Table
I.
For isoenzymes GI and GII the k/K
parameter
increased 2 orders of magnitude from laminarihexaose (DP 6) to
laminariheptaose (DP 7) and one order of magnitude for isoenzyme GIII (). When these data are presented graphically (Fig. 2), the relatively steady increase in k
/K
for isoenzyme
GIII can be more easily seen.
Figure 2:
Dependence
of rate parameters k/K on DP of
laminaridextrins. GI, GII, and GIII denote
1,3-
-glucanase isoenzymes GI, GII, and GIII,
respectively.
The k/K
values
obtained for each isoenzyme during hydrolysis of substrates of DP
7-9 are consistent with results reported when the isoenzymes
hydrolyze laminarin (DP approximately 25); again isoenzyme GIII had a
lower specific activity and k
/K
catalytic
efficiency (or specificity) factor than the other two isoenzymes
(Hrmova and Fincher, 1993).
Bond-Cleavage Frequencies
The bond-cleavage
frequencies of laminaridextrins of DP 2-9 are shown
diagrammatically in Fig. 3for isoenzymes GI, GII, and GIII. The
oligosaccharide substrates are drawn in their preferred binding
positions. It should be noted that short oligosaccharides might also
bind, unproductively, at subsites some distance from the catalytic
amino acids, but hydrolysis would not occur if there were no glycosidic
linkage spanning the catalytic site (Chen et al., 1995). The
observation that the preferred binding position of the octasaccharide
does not involve all the eight subsites in isoenzymes GI and GII can
probably be attributed to subtle differences in conformational freedom
between internal glycosyl residues and those at the nonreducing or
reducing ends of the substrate (MacGregor et al., 1994), and
it is possible that these differences are recognized at the
glucosyl-binding, subsite level.
Figure 3:
Schematic models of productive
enzyme-substrate complexes (positional isomers) and bond-cleavage
frequencies of 1-H-labeled laminaridextrins of DP 2-9
during hydrolysis with 1,3-
-glucanase isoenzymes GI (A),
GII (B), and GIII (C). Symbols used:
, D-glucopyranosyl residue;
&cjs0604;&cjs0604;,
1-
H-labeled reducing end glucosyl residue; --,
1,3-
-glucosidic linkage. The numbers above the glycosidic linkages
indicate bond-cleavage frequencies calculated as described by Allen and
Thoma (1976). The arrow shows the position of catalytic amino
acids.
The numerical values in Fig. 3reflect the cleavage frequency of individual glycosidic
linkages of the oligosaccharide substrate on the enzyme surface. Thus,
in Fig. 3A the bond-cleavage frequencies for
laminaripentaose (DP 5) show that the glycosidic linkage adjacent to
the nonreducing terminus is never cleaved (bond-cleavage frequency
= 0) and that in approximately 5% of substrate-enzyme encounters
(bond-cleavage frequency = 0.046) the second glycosidic linkage
from the nonreducing terminus is hydrolyzed; in 79% of substrate-enzyme
encounters (bond-cleavage frequency = 0.785), the third linkage
from the nonreducing terminus is hydrolyzed; and in 17% of encounters,
(frequency = 0.169) the linkage adjacent to the reducing
terminal residue is cleaved. An example showing the calculation of
bond-cleavage frequency of the second glycosidic linkage from the
nonreducing terminus of laminariheptaose (DP 7) by isoenzyme GII is
shown in Fig. 4. The gradient of the curve in Fig. 4(0.9338) corresponds to the bond-cleavage frequency of the
corresponding glycosidic linkage shown in Fig. 3B.
Figure 4:
Bond-cleavage frequency determination of
laminariheptaose (DP 7) during hydrolysis by 1,3--glucanase
isoenzyme GII. The slope of the line gives the bond-cleavage frequency
for the second glycosidic linkage from the nonreducing terminus; its
cleavage releases [
H]laminaripentaose (DP 5). The
conditions of hydrolysis are specified in Table
I.
Comparisons of the bond-cleavage frequencies of laminaridextrins
reveal small but significant differences between the three isoenzymes,
in particular with respect to the preferred binding positions of
oligosaccharides of DP 4 and 8 (Fig. 3). The appearance of some
products with DPs higher than 2 during the prolonged incubation of the
enzymes with laminaribiose indicated that transglycosylation reactions
occurred (data not shown).
Subsite Binding Energies
Pairs of positional
isomers of enzyme-substrate complexes used in calculating subsite
binding affinities of the 1,3--glucanase isoenzyme GI, together
with the affinities themselves, are given in I. Binding
affinities of the three isoenzymes are compared in Fig. 5. The bars show standard deviations for the calculations (Fig. 5). Although hydrolytic rates of unlabeled oligosaccharides
suggested that the barley 1,3-
-glucanases probably have between 7
and 10 binding subsites, the data did not allow unequivocal conclusions
regarding the number of subsites. However, calculations of binding
energies, as seen in Fig. 5and I, suggest that
there are eight binding subsites in each enzyme. Product analyses show
that catalytic amino acids are positioned between subsites 3 and 4 from
the nonreducing terminus of the substrates. We prefer to number the
subsites -3 to +5, to indicate their positions with respect
to the catalytic site (I, Fig. 5). The absence of
any significant binding at positions beyond -3 and +5 (I, Fig. 5) indicates that the binding sites of the
enzymes are restricted to eight glucosyl-binding subsites. Binding
energies have been expressed in the literature either as positive or
negative values (Allen and Thoma, 1976; Suganuma et al., 1978;
Biely et al., 1991). Here, we use positive values to indicate
binding; negative values therefore indicate repulsion between the
enzyme and the glycosyl residue aligned with the particular subsite and
values near zero indicate that no binding occurs.
Figure 5:
Subsite
maps of 1,3--glucanase isoenzymes GI (A), GII (B), and GIII (C). Subsite binding affinities (A
) are shown for individual glucosyl residues
bound to the enzyme. The number of subsites and the position of the
catalytic sites were determined according to Suganuma et al. (1978). Values shown for subsites -1 and +1 are
calculated by dividing the total binding energy of the subsites by two
(see ``Results''). The arrow shows the position of
catalytic amino acids. The error bars indicate standard
deviations, which were calculated from data for pairs of positional
isomers of enzyme-substrate complexes. In some cases, only one pair of
positional isomers will bind at a particular subsite, and standard
deviations could not be calculated in these
cases.
Theoretical
constraints preclude the accurate calculation of binding energies at
subsites -1 and +1, which are jointly estimated from the K of an oligosaccharide that is longer
than the entire substrate-binding region (Suganuma et al.,
1978). Typical Lineweaver-Burk plots used for the determination of K
values of laminarinonaose (DP 9) are
presented in Fig. 6. Values for K
of 0.646 mM, 0.777 mM, and 1.332 mM for isoenzymes GI, GII, and GIII, respectively, were used to
calculate the sum of the binding energies at subsites -1 and
+1. There has been considerable debate as to how these binding
values around the catalytic site should be presented and, indeed,
whether they should be calculated at all (Thoma and Allen, 1976;
Suganuma et al., 1978; Biely et al., 1983). Although
the binding energies of subsites -1 and +1 are often
represented in histograms of the type shown in Fig. 5as a single
energy value that spans the catalytic site (Suganuma et al.,
1978; Biely et al., 1981; Matsui et al., 1991;
Ajandouz et al., 1992;), we have chosen to divide the total
binding energy for the region by two and distribute it evenly across
the positions corresponding to glucosyl residues -1 and +1 (Fig. 5). We acknowledge that there may be no direct way to
calculate the contribution to each of these two subsites (Allen, 1980),
but have partitioned the binding equally between the subsites, as
described above, to emphasize that there are two glucosyl binding
subsites in the active site of the enzyme in this region.
Figure 6:
Lineweaver-Burk plots for the hydrolysis
of laminarinonaose (DP 9) by 1,3--glucanase isoenzymes GI, GII,
and GIII. Kinetic data were processed with a nonlinear regression
analysis based on Michaelis-Menten kinetics. Units for reaction rate (v) are µmol of laminarinonaose hydrolyzed per min/nmol of
enzyme and for substrate concentration (S) are
mM.
/K
as a function
of chain length (Thoma and Allen, 1976; Suganuma et al.,
1978). The determination of bond-cleavage frequencies uses product
analysis to define the disposition of the catalytic amino acids within
the active site, but depends on the availability of asymmetrically
labeled oligomeric substrates. Oligosaccharides can be labeled at their
reducing terminal glycosyl residues by reduction with radioactive or
nonradioactive sodium borohydride (Cole and King, 1964; Wong et
al., 1977; Bray and Clarke, 1992) or p-nitrophenyl
derivatives can be used (Ajandouz et al., 1992; MacGregor et al., 1992). To minimize any perturbations in
oligosaccharide structure (MacGregor et al., 1994), tritiation
of reducing terminal residues by tritium gas exchange (Evans et
al., 1974) is the preferred procedure (Biely et al.,
1981).
/K
, which
represents the association constant for a productive enzyme-substrate
complex and which can be used with the product analysis data to
calculate individual subsite binding affinities (Suganuma et
al., 1978). However, care must be exercised in kinetic analyses of
polysaccharide endohydrolases, because these enzymes can catalyze
bimolecular reactions such as transglycosylation or condensation
reactions. In addition, multiple hydrolytic events (``multiple
attack'') can occur during a single enzyme-substrate encounter.
Finally, the products of one reaction can become substrates in
subsequent reactions or a new substrate molecule can bind before one of
the original products dissociates from the enzyme, whereby a ternary
enzyme-substrate complex can form (Biely et al., 1991). These
effects can be minimized by using low substrate concentrations and by
rigorous adherence to the measurement of initial reaction rates.
-glucan
endohydrolases from barley seedlings. The three isoenzymes each have
eight
-glucosyl-binding subsites and the catalytic amino acids are
positioned to hydrolyze the glycosidic linkage that lies between the
third and fourth glucosyl residue from the nonreducing terminus of the
bound substrate (Fig. 5). Crystallographic data show that barley
1,3-
-glucanase isoenzyme GII folds to give an
/
barrel
structure, with a deep cleft approximately 40 Å long running over
the surface of the molecule (Chen et al., 1993b; Varghese et al., 1994). The length of the cleft, which clearly
represents the substrate-binding region of the enzyme, is sufficient to
accommodate 7 or 8 residues of an extended 1,3-
-glucan chain
(Varghese et al., 1994) and the two catalytic glutamic acid
residues, Glu
and Glu
(Chen et
al., 1993a), lie about one-third of the way down the cleft
(Varghese et al., 1994). Furthermore, structural studies of
the enzyme with an inhibitor bound in the substrate-binding cleft show
that the catalytic amino acids are asymmetrically placed, toward the
nonreducing end of the bound substrate (Chen et al., 1995).
Thus, the data obtained here for the number of binding subsites and the
location of the catalytic site along the substrate-binding region of
barley 1,3-
-glucanase isoenzyme GII (Fig. 3B and
5B) are consistent with the three-dimensional data generated
by x-ray crystallography (Chen et al., 1995; Varghese et
al., 1994). A molecular model based on x-ray crystallographic data
(Varghese et al., 1994), in which an oligosaccharide occupies
the likely positions of subsites +2 to +5 in the substrate
binding cleft of isoenzyme GII, is shown in Fig. 7. The substrate
appears to fit into the cleft edgewise and no obvious helical
conformation of the type observed in long 1,3-
-glucan chains
(Stone and Clarke, 1993) can be seen (Fig. 7). The model does not
show the binding at subsites close to the catalytic amino acid residues (Fig. 7). Although binding patterns at subsites -2 to
+1 have been presented by Chen et al.(1995), these data
were based on studies with covalently bound inhibitors and until the
enzyme has been co-crystallized with native substrate, we cannot be
certain of the precise positions of glucosyl residues around the active
site.
Figure 7:
Molecular model of laminaritetraose (DP 4)
in the likely position of subsites +2 to +5 of the substrate
binding cleft of 1,3--glucanase isoenzyme GII; the reducing end of
the oligosaccharide lies to the right. The enzyme is oriented
with the
-barrel axis approximately vertical and with subsite
+5 over amino acid residue 193. The molecular surface of the
protein is shown in blue, and the oligosaccharide is
represented as a wire model, where carbon atoms are yellow,
oxygen atoms are orange, and C(O)-6 atoms are red.
Binding energies at individual subsites are generally similar
for the three isoenzymes, although some differences are observed (Fig. 5). For example, the binding affinity at subsite -3
is notably higher for isoenzyme GIII than for isoenzymes GI and GII.
The probable location of this subsite is on top of amino acid residue
34 and next to residues 56-58; isoenzyme GIII differs from
isoenzymes GI and GII at these residues (Xu et al., 1992).
High positive binding energies at subsites -2, +4, and
+5 indicate that -glucosyl residues in the substrate are
bound relatively tightly at these subsites. Crystallographic studies
show that there is a disaccharide binding site near Ala
of the 1,3-
-glucanase isoenzyme GII (Chen et al.,
1995); the disaccharide lies deep in the substrate binding cleft and
its position is entirely consistent with it occupying subsites +4
and +5. The negative binding energy at subsite +3 suggests
that glucosyl binding at this position is thermodynamically unfavorable
and although we can offer no functional explanation for this unexpected
result, similar effects have been noted in certain starch-degrading
enzymes (Suganuma et al., 1978, 1991; Matsui et al.,
1991). Because Met
forms a ridge across the substrate
binding cleft in this position, the glucosyl residue occupying subsite
+3 would not sit deep in the cleft. Rather, it would be relatively
exposed on the enzyme surface and its C(O)-6 atom would be pointing
outwards (Fig. 7). This could permit branching or substitution at
the C(O)-6 atom and the negative binding energy may be a consequence of
exposing the hydrophobic faces of the glucosyl ring.
-glucan molecule is
probably bound initially at subsites toward each end of the
substrate-binding cleft. Tight binding of glucosyl residues near the
cleavage point, at subsites -2 and +2, might cause substrate
distortion at the catalytic site and thereby contribute to catalytic
efficiency (Thoma et al., 1971). However, as mentioned above,
a precise description of substrate binding by the 1,3-
-glucanases
awaits crystallographic data from enzyme-substrate complexes. Such data
would also lead to the identification of amino acids that mediate the
binding of glucosyl residues at each of the eight subsites, and the
relative importance of individual amino acids in substrate binding
could be confirmed by site-directed mutagenesis (Moreau et
al., 1994).
-glucanases isoenzymes from barley. The three isoenzymes have
almost certainly evolved from a common ancestral enzyme, and although
considerable diversification has occurred in their primary structures
(Xu et al., 1992; Hand Fincher, 1995), they each
bind eight glucosyl residues of the substrate, the disposition of
catalytic amino acids with respect to the binding subsites is
conserved, and subsite binding energies are very similar in the three
isoenzymes. The observation that the enzymes bind relatively long
regions of their 1,3-
-glucan substrate raises questions as to the
efficiency with which they can perform their proposed function in
hydrolyzing fungal cell wall
-glucans during plant-microbe
interactions (Hrmova and Fincher, 1993). The fungal wall
1,3-
-glucan would require relatively long unsubstituted or
unbranched domains if maximal rates of hydrolysis were to be achieved.
Alternatively, the C(O)-6 atoms of the 1,3-
-glucan chain, which
carry the glucosyl substituents or polymeric side chains, would need to
project out of the substrate-binding cleft to prevent steric hindrance
during substrate binding. Although it appears likely that many C(O)-6
atoms of the bound 1,3-
-glucan would be buried in the substrate
binding cleft, at subsite +3 and possibly at subsite +5 the
methanolic group may project into the solvent, and this could allow
branching or substitution of the corresponding glycosyl residues (Fig. 7). A thorough understanding of all these substrate-enzyme
interactions at the molecular level could well present opportunities
for the use of protein engineering to improve catalytic efficiency or
broaden the specificity of plant 1,3-
-glucanases and thereby to
enhance their ability to rapidly hydrolyze cell wall
1,3;1,6-
-glucans from a broad range of fungal pathogens.
Table: Experimental conditions for kinetic analyses of
laminaridextrins of DP 2-9 during hydrolysis by barley
1,3--glucanase isoenzymes GI, GII, and GIII
Table: Kinetic parameters,
k/K
, for 1,3-
-glucanase isoenzymes GI,
GII, and GIII during hydrolysis of laminaridextrins of DP 2-9
Table: Subsite
binding affinities (A) of 1,3-
-glucanase isoenzyme GI
is likely to be the catalytic acid in the
barley 1,3-
-glucanase isoenzyme GII described here, rather than
Glu
as suggested by Chen et al. (1993a).
3)-
-Glucans, La Trobe University Press, Bundoora, Victoria, Australia
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.