©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subsite Affinities and Disposition of Catalytic Amino Acids in the Substrate-binding Region of Barley 1,3--Glucanases
IMPLICATIONS IN PLANT-PATHOGEN INTERACTIONS (*)

Maria Hrmova (1), Thomas P. J. Garrett (2), Geoffrey B. Fincher (1)(§)

From the (1)Department of Plant Science, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia and the (2)Biomolecular Research Institute, 343 Royal Parade, Parkville, Victoria 3052, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Oligo-1,3--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.


INTRODUCTION

The 1,3--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).

These possibilities suggest that a detailed knowledge of substrate specificities of the 1,3--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.

The three-dimensional structure of barley 1,3--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.


MATERIALS AND METHODS

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 equivalentsmin 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 cmh at 20-22 °C. Eluted carbohydrate was detected using the orcinol-HSO 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/HO (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 ENHANCE 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 TBqmol, which corresponded to 80-465 Cimol. 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.

To confirm that tritium was introduced only into the reducing terminal glucosyl residues, the oligosaccharides were reduced with NaBH (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.

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--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.

In the calculation of the kinetic parameter, k/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,

On-line formulae not verified for accuracy

where [S] and [S] represent substrate concentrations at zero time and after enzyme addition, respectively. By plotting the function,

On-line formulae not verified for accuracy

where G 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).

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 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,

On-line formulae not verified for accuracy

and for these calculations we used bond-cleavage frequencies 0.013. Standard deviations were calculated according to Samuels(1991) by taking into account data from pairs of positional isomers of enzyme-substrate complexes.

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 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 TBqmol 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).


RESULTS

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.

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/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/Kfor 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/Kcatalytic 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.




DISCUSSION

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/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).

The kinetic analyses allow the determination of the parameter k/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.

Against this theoretical background we have used subsite mapping to describe enzyme-substrate binding in three 1,3--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.

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--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).

In summary, we have used subsite mapping procedures to describe certain aspects of substrate-binding in three 1,3--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



FOOTNOTES

*
This work was supported by grants from the Australian Research Council (to G. B. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 61-8-303-7296; Fax: 61-8-303-7109.

The abbreviation used is: DP, degree of polymerization.


ACKNOWLEDGEMENTS

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 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).


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