Structural Advantage of Sugar Beet α-Glucosidase to Stabilize the Michaelis Complex with Long-chain Substrate

Background: Most plant α-glucosidases prefer long-chain substrates.

Results: Inhibitory and structural analyses using the unique 4–10-mer inhibitors identified the substrate binding mode of the enzyme far from the active-site pocket.

Conclusion: The structure of the substrate-binding subsites was suitable for single helical conformation of amylose.

Significance: The enzyme seems to ingeniously use the self-stabilizing property of the substrate to form a stable ES complex.

Abstract

The α-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower K_m and higher k_cat/K_m for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligosaccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of α-glycosidases. The clear correlation obtained between log K_i of the acarviosyl-maltooligosaccharides and log(K_m/k_cat) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the K_m for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates.

Introduction

Glucans of various types are widely distributed in nature. Each type of glucan adopts a unique conformation, which is dependent on the type of glucosidic linkage in the molecule. For the effective degradation of a variety of glucans, various subsites are present in glycoside hydrolases (1). Most endo-type glycoside hydrolases such as α-amylase, dextranase, and cellulase contain cleft-shaped subsites (2–5), which, in β-amylase, extend from a pocket-shaped active site (6). Cellobiohydrolase, a cellulose-hydrolyzing enzyme, binds the β-1,4-glucan chain within a tunnel-shaped subsite (7). In the case of Coprinopsis cinerea cellobiohydrolase, the conformation of the tunnel-shaped subsite has been observed to change from an open to closed conformation in response to substrate binding (8). Such structures of enzyme subsites facilitate the loosening of the packed conformation of glucans via multiple interactions and contribute to the effective degradation of these carbohydrate polymers.

α-Glucosidase is an exo-type enzyme, which catalyzes the hydrolysis of α-glucosidic linkage at the non-reducing termini of substrate molecules. In addition to other exo-type glycosidases, α-glucosidase has a pocket-shaped active site. A majority of α-glucosidases exhibits preference for disaccharides and trisaccharides as substrates (9, 10). In contrast, several α-glucosidases belonging to glycoside hydrolase family 31 (GH31)⁴ (11) are known to display specificity for substrates with a high degree of polymerization (DP) (12–14). Among them, the α-glucosidase from sugar beet exhibits the highest specificity for long-chain maltooligosaccharides and soluble starch due to low K_m and high k_cat/K_m (15). The crystal structure of SBG in a complex with the pseudo-tetrasaccharide inhibitor, acarbose (AC4), was determined for structural analysis of its substrate specificity (16). The overall structure of SBG was found to be substantially similar to that of the other GH31 α-glucosidases of known structure, and comprises a catalytic domain with (β/α)₈-barrel fold, and the N- and C-terminal domains with β-sandwich structures. SBG has a pocket-shaped active site, also found in other related α-glucosidases (9, 10, 17–19). This pocket is formed by loops that exist between the β-strands and the α-helices of the catalytic domain as well as a long loop (designated as N-loop) that protrudes from the N-terminal domain. The loops that follow the third and fourth β-strands of the catalytic domain contain short insertions named subdomains b1 and b2, respectively. The structural study (16) demonstrated that Phe²³⁶ and Asn²³⁷ on the N-loop play a role in the specificity of SBG for long-chain substrates. These residues are involved in substrate binding at subsites +2 and +3, and direct the reducing end of the substrate toward subdomain b2, where Ser⁴⁹⁷ is poised to bind the substrate at subsite +4 (16, 20).

The aforementioned studies provided important clues toward understanding the reason behind the specificities of SBG and the other GH31 enzymes for longer substrates; however, a complete understanding is yet to be achieved, particularly because of the binding of substrates at subsites remote from the active site. In the present study, to comprehend the binding of long-chain substrates to SBG, a series of unique long-chain inhibitors, acarviosyl-maltooligosaccharides (AC5-AC10, where the numeral represents DP), was employed as the ligands for structural resolution of the resulting complexes. The acarviosyl-maltooligosaccharides have longer maltooligosaccharide parts than the maltose unit of AC4 (Fig. 1), and were synthesized from commercially available AC4 and maltooligosaccharides with a disproportionating enzyme (21); this methodology resulted in the conversion of more than half of the AC4 into acarviosyl-maltooligosaccharides.

FIGURE 1.

Acarviosyl-maltohexaose (AC8).

In the current study, the potential of the various acarviosyl-maltooligosaccharides for the inhibition of SBG was evaluated, and the crystal structures of SBG bound with AC5−AC8 were determined. The structures of the complexes of SBG with acarviosyl-maltooligosaccharides elucidated the mechanism of substrate binding at subsites remote from the active site pocket. The specificity of SBG for long-chain substrates is likely due to the structure of remote subsites, which accommodate the stable single-helical conformation of long-chain amylose.

EXPERIMENTAL PROCEDURES

Materials

The substrates for kinetic analysis included a series of maltooligosaccharides ranging from maltose (Glc₂) to maltoheptaose (Glc₇) (Nihon Shokuhin Kako, Tokyo, Japan), amylose EX-I (Glc₁₈, average DP of 18; Hayashibara, Okayama, Japan), and soluble starch (Nacalai Tesque, Kyoto, Japan). The concentration of non-reducing termini of the soluble starch was estimated as 0.136 μmol/mg using the Smith degradation method (22). A series of acarviosyl-maltooligosaccharides ranging from acarviosyl-maltotriose (AC5) to acarviosyl-maltooctaose (AC10) were enzymatically synthesized and purified by HPLC as previously reported (21). The purified acarviosyl-maltooligosaccharides were evaporated to dryness and dissolved in water, and their concentrations were determined on the basis of absorbance at 214 nm. For the nomenclature of the sugar rings of acarviosyl-maltooligosaccharides, the valienamine residue at the non-reducing end was termed as ring A, the 4-amino-4,6-dideoxy α-d-glucose residue as ring B, and the glucose residues in the maltooligosaccharyl moiety from the non-reducing toward the reducing side, as rings C, D, E, and so forth (Fig. 1).

Enzyme Purification and Preparation

Purification of native SBG from sugar beet seeds and preparation of endoglycosidase-F3-treated native SBG for crystallization were reported previously (16).

Heterologous expression of mutant SBGs in Pichia pastoris and their purification were carried out according to a previous report (20). The expression vector for each mutant enzyme was constructed by PCR using the PrimeSTAR mutagenesis basal kit (Takara Bio, Otsu, Japan), pGAPZαA vector carrying a SBG gene as the template, and primers 5′-AGGGACGCTAACTTGTATGGATCCCAC-3′ and 5′-CAAGTTAGCGTCCCTATTGAAGCTAGC-3′ for Leu²⁴⁰ → Ala mutation, 5′-CCATATGCTATCAATAATTCTGGAGGC-3′ and 5′-ATTGATAGCATATGGTGGATTGTCAAG-3′ for Lys⁴⁹³ → Ala mutation, 5′-GGCCGTCGTCCAATAAATAGCAAGACT-3′ and 5′-TATTGGACGACGGCCTCCAGAATTATT-3′ for Val⁵⁰¹ → Arg mutation, 5′-GGCCGTGCTCCAATAAATAGCAAGACT-3′ and 5′-TATTGGAGCACGGCCTCCAGAATTATT-3′ for Val⁵⁰¹ → Ala mutation, and 5′-CGTGTAGCTATAAATAGCAAGACTATT-3′ and 5′-ATTTATAGCTACACGGCCTCCAGAATT-3′ for Pro⁵⁰² → Ala mutation with the underlined nucleotides indicating the mutated codons.

Crystal Structure Analyses

Co-crystallizations of endoglycosidase F3-treated native SBG individually with AC5-AC8 were performed by the hanging-drop vapor diffusion method at 25 °C, with the following composition of the drops: 3 μl of the enzyme (4.2 mg/ml), 3 μl of reservoir solution (50 mm sodium acetate buffer (pH 4.5), 50 mm ammonium sulfate, and 16–18% PEG monomethyl ether 2000), and 1 μl of the ligand (33.3 mm AC5, 15.8 mm AC6, 7.79 mm AC7, or 4.44 mm AC8). X-ray diffraction data were collected on beamline BL41XU at SPring-8 (Hyogo, Japan) in the same manner as described previously (16). All diffraction data sets were indexed, integrated, scaled, and merged using XDS (23). Crystals of AC5 and AC8 complex belonged to the P2₁2₁2₁ space group and are isomorphous with crystal of AC4 complex, whereas crystals of AC6 and AC7 complex belonged to the I222 space group. Complex structures of AC5 and AC8 were determined by a molecular replacement method with phenix.automr (24, 25) using the SBG-AC4 complex (Protein Data Bank code 3W37) as a search model and those of AC6 and AC7 were determined by rigid body refinement with phenix.refine (26) using the SBG-AC4 complex model. After several cycles of manual model corrections with Coot (27) and refinement with REFMAC5 (28) and phenix.refine, the refinement converged. The coordinates and structure factors have been deposited in the Protein Data Bank with codes 3WEL, 3WEM, 3WEN, and 3WEO for the AC5, AC6, AC7, and AC8 complexes, respectively. Data collection and refinement statistics are summarized in Table 1.

TABLE 1.

Data collection and refinement statistics for data sets of SBG complex forms

Crystal	AC5 complex	AC6 complex	AC7 complex	AC8 complex
Data collection
Space group	P212121	I222	I222	P212121
Unit cell parameters
(a, b, c) (Å)	(84.7, 97.9, 106.8)	(97.4, 139.4, 149.4)	(97.4, 138.9, 149.0)	(86.2, 99.2, 107.4)
Resolution rangea (Å)	38.9–1.84 (1.95–1.84)	44.4–2.59 (2.75–2.59)	44.2–2.59 (2.75–2.59)	43.0–1.45 (1.54–1.45)
No. of unique reflectionsa	77,140 (11,929)	31,809 (4,956)	31,018 (4,862)	162,274 (25,946)
Rmeasa	0.116 (0.788)	0.118 (0.731)	0.114 (0.710)	0.118 (0.586)
Completenessa (%)	98.8 (95.8)	99.4 (97.3)	97.4 (95.5)	99.5 (99.3)
〈I/σ(I)〉a	9.16 (2.01)	16.87 (3.14)	15.04 (3.22)	8.02 (2.46)
Multiplicitya	3.55 (3.55)	5.57 (5.49)	5.61 (5.50)	3.66 (3.61)
Refinement
Rwork	0.1724	0.2038	0.1993	0.1234
Rfree	0.1975	0.2374	0.2451	0.1499
No. of protein atoms	6726	6606	6606	6824
No. of water molecules	604	216	175	1027
No. of sugar residues of N-glycans	6	5	5	6
Root mean square deviation values from ideal
Bond lengths (Å)	0.007	0.005	0.003	0.008
Bond angles (°)	1.140	1.182	0.799	1.308
Ramachandran plot analysis
Favored region (%)	96.8	97.6	96.1	97.5
Allowed region (%)	3.0	2.3	3.7	2.4
Outlier region (%)	0.2	0.1	0.2	0.1

^a Values in parentheses are for the highest-resolution shell.

Biochemical Assays

α-Glucosidase activity, protein concentration, and the effects of pH were measured as described previously (20). The type of inhibition and inhibition constants (K_i) of AC4-AC10 for native SBG were determined using 1/[S] versus 1/v plots. The hydrolysis velocities for Glc₇ (0.2, 0.24, 0.3, 0.4, 0.6, and 1.2 mm) in the presence of AC4 (6, 8, and 10 μm), AC5 (2, 4, and 6 μm), AC6 (0.5, 1, and 2 μm), AC7 (0.5, 1, and 2 μm), AC8 (0.5, 1, and 2 μm), AC9 (0.5, 1, and 2 μm), or AC10 (0.5, 1, and 2 μm) were measured under standard reaction conditions (20). The values of apparent K_m for Glc₇ were obtained from the x axis intercept of 1/[S] versus 1/v plots in the presence of the inhibitor, and the values of K_i (μm) were calculated from the equation,

where K_m^app is the apparent K_m in the presence of the inhibitor, K_m is the actual K_m in the absence of inhibitor, and [I], the concentration of the inhibitor. The values of mean ± S.D. for K_i at three inhibitor concentrations were calculated.

For the determination of kinetic parameters (k_cat, K_m, and k_cat/K_m), the initial rates for eight substrate concentrations were measured, and the kinetic parameters k_cat (s⁻¹) and K_m (mm) were determined from [S] versus v plots by fitting to Michaelis-Menten equation. The enzyme concentrations used were 0.388–0.778 (native SBG), 1.45–2.90 (L240A), 1.25–2.50 (K493A), 0.944–1.89 (V501A), 1.11–2.22 (V501R), or 1.41–2.81 nm (P502A).

The correlations between log K_i for AC4−AC7 and log(K_m/k_cat) for the hydrolysis of Glc₄−Glc₇ or between log K_i for AC4−AC7 and log K_m for Glc₄−Glc₇ were analyzed for evaluating the transition state mimicry of the acarviosyl-maltooligosaccharides. The correlations were derived on the basis of the equation K_i = dK_TS = dk_non(K_m/k_cat), where d and k_non represent proportionality and non-enzymatic reaction rate constants, respectively (29).

RESULTS

SBG Inhibition by Acarviosyl-maltooligosaccharides

The type of inhibition and the inhibition constants of AC4-AC10 for the hydrolysis of native SBG, which was prepared from sugar beet seeds, were evaluated from 1/[S] versus 1/v plots using Glc₇ as a substrate. The 1/[S] versus 1/v plots of all the acarviosyl-maltooligosaccharides showed linear correlation and intersected with each other on the y axis, indicating that the acarviosyl-maltooligosaccharides are competitive inhibitors of SBG. AC4 inhibited SBG with K_i of 15.4 ± 3.5 μm, and the K_i values decreased with increasing DP of the inhibitor (Table 2). In particular, a greater difference was found between the K_i of AC4 and AC5 compared with the other acarviosyl-maltooligosaccharides. The plots of log K_i for AC4−AC7 against log(K_m/k_cat) for the hydrolysis of Glc₄−Glc₇ as well as log K_i for AC4−AC7 against log K_m for Glc₄−Glc₇ showed linear correlation with correlation coefficients of r = 0.964 (slope = 1.44) and r = 0.985 (slope = 1.59), respectively (Fig. 2). These results suggest that the acarviosyl-maltooligosaccharides mimic both the transition and ground states.

TABLE 2.

Kinetic parameters for the substrates and inhibition constants of AC4-AC10 for the reaction of the native SBG

Substrate	Kinetic parametersa			Inhibition constants
	kcat	Km	kcat/Km	Inhibitor	Kib	Type
					μm
Glc2	384 ± 4	17.8 ± 0.9	21.6
Glc3	442 ± 2	2.91 ± 0.07	152
Glc4	388 ± 5	1.74 ± 0.03	223	AC4	15.4 ± 3.5	Competitive
Glc5	478 ± 2	0.623 ± 0.017	767	AC5	2.66 ± 0.21	Competitive
Glc6	464 ± 2	0.359 ± 0.004	1,290	AC6	1.63 ± 0.73	Competitive
Glc7	457 ± 1	0.327 ± 0.002	1,400	AC7	0.845 ± 0.160	Competitive
Glc18	436 ± 4	0.293 ± 0.010	1,490	AC8	0.888 ± 0.032	Competitive
Soluble starchc	405 ± 3	0.166 ± 0.002	2,440	AC9	0.936 ± 0.177	Competitive
				AC10	1.01 ± 0.13	Competitive

^a k_cat, s⁻¹; K_m, mm; k_cat/K_m, s⁻¹ mm⁻¹. Each k_cat or K_m with standard deviation is the average value of the triplicate measurements.

^b Each K_i with standard deviation is average value at three concentrations of inhibitor.

^c The K_m value for soluble starch is its concentration of non-reducing termini.

FIGURE 2.

Linear correlations between log K_i and log(K_m/k_cat), and between log K_i and log K_m. Native SBG was employed for determining K_i of AC4–AC7 and the kinetic parameters for the hydrolysis of Glc₄–Glc₇. Regression lines along with correlation coefficient (r) are shown.

Crystal Structures of Complexes of SBG with Acarviosyl-maltooligosaccharides

SBG was co-crystallized individually with the acarviosyl-maltooligosaccharides AC5, AC6, AC7, and AC8, and the structures of the corresponding complexes were determined at 1.8, 2.6, 2.6, and 1.5 Å, respectively (Table 1). Analysis of the structures of all these complexes revealed that SBG existed as a monomer in each asymmetric unit and had three N-glycans at Asn⁴⁰⁴, Asn⁷²⁸, and Asn⁸²³ (Fig. 3A), as well as the previous crystal structures of SBG (16). The overall structures obtained in the present study were almost identical to the ligand-free and AC4-complex structures, and the root mean square deviations between every pair, as estimated by the Dali pairwise server (30), was within 0.5 Å. Co-crystals of sufficient size for structure determination could not be obtained for SBG with AC9 or AC10.

FIGURE 3.

Binding of acarviosyl-maltooligosaccharides to SBG. A, overall structure of SBG bound with AC8 (purple stick). The active site pocket comprises the N-loop protruding from the N-terminal domain (pink), the catalytic (β/α)₈-barrel domain (light orange), and subdomains b1 (orange) and b2 (yellow), which are inserted into the catalytic domain. The proximal and distal C-terminal domains (light green and light blue) are not directly involved in substrate binding. N-Glycans (green stick) were attached to Asn⁴⁰⁴, Asn⁷²⁸, and Asn⁸²³. B, the σ_A-weighted composite omit maps (2F_o − F_c) are drawn around AC5-AC8 molecules and contoured at 1σ. C, superimposition of AC4 (cyan), AC5 (green), AC6 (magenta), AC7 (yellow), and AC8 (purple) bound to SBG.

The electron density corresponding to acarviosyl-maltooligosaccharides was observed only at the active site of each structure (Fig. 3B). The electron density of the reducing glucose residue of AC7 (ring G) was not completely observed. This might be because of the kinetically weak affinity at subsite +6 (20) or the low-resolution structure. Fig. 3C shows the structural superposition of acarviosyl-maltooligosaccharides from all the complexes, including the previously determined structure of the AC4 complex. The conformations of the rings of acarviosyl-maltooligosaccharides were observed to be nearly identical in all the structures; however, the conformations of ring G showed a slight difference between the AC7 and AC8 complexes. Compared with AC7, ring G of AC8 was observed to lie closer to the enzyme in a manner dependent on the interactions between ring H and subdomain b2 (see below). The acarviosine unit (rings A and B) was buried in the active-site pocket, whereas the maltooligosaccharide part was bound to the N-loop and subdomain b2. All residues in the N-loop and subdomain b2 had identical conformations, although a slight difference was observed in the side chain of Lys⁴⁹³ in the structure of the AC8 complex (data not shown).

The conformations of rings A−C were extended, and these rings, particularly the acarviosine unit, were tightly bound to the enzyme through several hydrogen bonds. In contrast, the conformations of rings D–H were similar to that of the native helical conformation through intramolecular hydrogen bonds with adjacent glucose residues (Fig. 4A). All glucose residues had cis-orientation and were connected by O2′–O3 hydrogen bonds, for instance, O2 (ring D)–O3 (ring E); only the rings F and G were trans-oriented, and two hydrogen bonds, O6 (ring F)–O3 (ring G) and O5 (ring F)–O3 (ring G), were observed.

FIGURE 4.

Substrate binding to N-loop and subdomain b2. Top (A) and side (B) views focusing on the N-loop and subdomain b2 bound to AC8 are shown as stereo diagrams. Amino acid residues and water molecules interacting with AC8 are represented by a stick model and red sphere, respectively. Two catalytic residues are indicated by asterisks. The dashed lines indicate hydrogen bonds. Color coding is similar to Fig. 3A. C, multiple sequence alignment at a region spanning Lys⁴⁹³ to Pro⁵⁰² was produced by MUSCLE (36). SOG, spinach α-glucosidase (O04893); BWG, buckwheat α-glucosidase (H. Mori, unpublished data); ONG1, rice α-glucosidase isozyme 1 (Q653V4); ONG2, rice α-glucosidase isozyme 2 (Q653V7); BAG, barley α-glucosidase (Q43763).

Rings C, D, and E were bound to the N-loop and subdomain b2 (Fig. 4B). Phe²³⁶, Asn²³⁷, and Leu²⁴⁰ on the N-loop contacted rings D and E, and directed the subsequent glucose residues (rings F–H) toward subdomain b2. Phe²³⁶ and Asn²³⁷ established interactions with ring D, as shown in our previous reports (16, 20), whereas the side chain of Leu²⁴⁰ established van der Waals contact with ring E. Ring E also bound the enzyme through hydrogen bonds between the O6 and O5 atoms and the hydroxy group of Ser⁴⁹⁷ in subdomain b2. Rings F, G, and H were located at a region of subdomain b2 spanning Lys⁴⁹³ to Pro⁵⁰². The conformation of this region was tightly packed by 10 hydrogen bonds between the side chains and backbone, and this region interacted with rings F, G, and H via hydrogen bonds through the backbone and van der Waals contact through the side chains. Ring F interacted with the backbone carbonyls of Ser⁴⁹⁷ and Gly⁴⁹⁸ via a water molecule. Three hydrogen bonds were observed between ring H (O2 and axially oriented O1) and the backbone carbonyls of Arg⁵⁰⁰ and Gly⁴⁹⁹. Val⁵⁰¹ was significantly close to rings F and G, and Lys⁴⁹³ and Pro⁵⁰² were found in the vicinity of ring H. The multiple sequence alignment indicated that the region from Lys⁴⁹³ to Pro⁵⁰² was highly conserved but Val⁵⁰¹ of SBG was atypical among plant α-glucosidases (Fig. 4C).

Site-directed Mutagenesis

The structures of these complexes suggested that the side chains of Leu²⁴⁰, Lys⁴⁹³, Val⁵⁰¹, and Pro⁵⁰² are likely to be involved in substrate binding. The contributions of these residues to substrate binding were assessed using the mutant enzymes, L240A (Leu²⁴⁰ → Ala), K493A (Lys⁴⁹³ → Ala), V501A (Val⁵⁰¹ → Ala), and P502A (Pro⁵⁰² → Ala), which were generated using site-directed mutagenesis. In addition, the characterization of V501R (Val⁵⁰¹ → Arg) was also performed to determine the functional role of the conserved arginine residue in the other plant α-glucosidases (Fig. 4C). The optimum pH (pH 4.9) for Glc₂ hydrolysis and pH stability (pH 3.0–10.3) of all the mutant enzymes was nearly identical to those of the wild-type recombinant SBG (rSBG) (20). The kinetic properties were determined for a series of maltooligosaccharides, Glc₁₈, and soluble starch (Table 3). All recombinant enzymes including rSBG exhibited lower k_cat values than the native SBG. This may be caused by excessive glycosylation observed in the recombinant enzymes produced by P. pastoris.

TABLE 3.

Kinetic parameters^a of the SBG variants

^a k_cat, s⁻¹; K_m, mm; k_cat/K_m, s⁻¹ mm⁻¹. Each k_cat or K_m with standard deviation is the average value of the triplicate measurements.

^b The K_m value for soluble starch is its concentration of non-reducing termini.

Both K493A and P502A exhibited almost the same K_m values for all the substrates as rSBG, even though their k_cat values decreased. The k_cat/K_m values for soluble starch were 80- (K493A) and 88-fold (P502A) higher than each for Glc₂, namely the substrate specificities of the mutant enzymes were almost identical to rSBG.

L240A showed that its k_cat for all the substrates equaled 59–72% that of rSBG. In contrast, the extent of decrease in k_cat/K_m values was dependent on the DP of the substrates. For instance, k_cat/K_m for Glc₂-Glc₄ substrates equaled 51–56% that of rSBG, whereas for Glc₅-Glc₇, the values equaled only 23%. Reduction in k_cat/K_m was also observed for Glc₁₈ and soluble starch (21% of rSBG values for both substrates). This reduction in k_cat/K_m was associated with an increase in K_m. Significantly higher K_m of L240A was observed for substrates with DP of more than 4; for instance, the K_m for Glc₂–Glc₄ was 1.1–1.3-fold that of rSBG, whereas for Glc₅–Glc₇, it was 2.6–2.8-fold. These results indicated that the mutation (Leu²⁴⁰ → Ala) decreased the binding affinity in the transition state and the ES complex at subsite +4. This is consistent with structure-based analysis, which revealed that Leu²⁴⁰ established contacts with ring E of acarviosyl-maltooligosaccharides.

The changes in kinetic parameters of the V501A and V501R mutants paralleled those of L240A, with the k_cat values for all substrates were affected equally, but with greater reduction in k_cat/K_m values for substrates with DP of more than 4 as a consequence of 1.2–1.8-fold increased K_m for these substrates. These results suggest that the substitutions of Val⁵⁰¹ caused a reduction in affinity at subsite +4, which is in contradiction with the structure-based analysis, which revealed that Val⁵⁰¹ is located close to rings F and G, occupying subsites +5 and +6.

DISCUSSION

In the current study, acarviosyl-maltooligosaccharides were employed for clarifying the mode of substrate binding at subsites remote from the active site pocket. The K_i values of the acarviosyl-maltooligosaccharides AC5–AC10 for SBG were significantly lower compared with AC4, indicating that these acarviosyl-maltooligosaccharides are more effective inhibitors of SBG than AC4. The clear correlation observed between log K_i for AC4−AC7 and log(K_m/k_cat) for the hydrolysis of Glc₄−Glc₇ suggests that the inhibitors are transition state analogs (29). Relative to the substrate, the tighter binding of the acarviosyl-maltooligosaccharides to the active site results in a value of K_i, which is 3 orders of magnitude lower than K_m (upon equating K_m with K_s); this could be considered a consequence of the valienamine unit, which is considered to mimic the glycosyl cation-like transition state. The reduction in K_i with an increase in DP can be accounted for by a decrease in the dissociation constant of the maltooligosaccharide unit from the enzyme, because the correlation of log K_i versus log K_m is similar to that of log K_i versus log(K_m/k_cat). In the hydrolysis reaction, the increase in k_cat/K_m with an increase in the DP of substrates is due to the decrease in K_m for these substrates. The values of k_cat are almost unaltered, but K_m decreases with an increase in the DP of maltooligosaccharides from 2 to 7 (Table 2). In other words, maltose binding at subsites −1 and +1 provides sufficient binding energy for lowering the activation energy of SBG, and the binding energy at the subsequent subsites +2, +3, and so forth are chiefly employed for decreasing the dissociation constant. This kinetic behavior is very similar to the inhibitory behavior of the acarviosyl-maltooligosaccharides; therefore, the binding of the acarviosyl inhibitors could be considered to represent the binding mode of the substrates, and hence, serves as an adequate probe for characterization of the remote substrate-binding site. The observed slope (1.44) of the correlation between log K_i and log(K_m/k_cat) as opposed to the expected slope (1.0) suggests a less than ideal mimicry of the transition state by the analog (31). The optimal mimicry of the transition state by an inhibitor should result in a 10⁻⁵-fold or lower K_i, which is described as K_TS in the case of transition state analog, compared with the K_m, considering the equation K_TS = k_non(K_m/k_cat) and the reported values of k_non/k_cat (29, 32). The evaluated K_i of the acarviosyl-maltooligosaccharides, of the range ((2.5−8.8) × 10⁻³)-fold that of the K_m for the substrate, indicates a lesser degree of mimicry despite the tighter binding of acarviosyl-maltooligosaccharides to the active site compared with the substrate. The slightly higher values of K_i of acarviosyl-maltooligosaccharides compared with the theoretical values are attributable to imperfect mimicry by the valienamine unit.

Crystal structure analyses of SBG bound with the acarviosyl-maltooligosaccharides revealed the molecular basis of substrate binding at a site distant from the active site pocket. The N-loop and the region spanning Lys⁴⁹³ to Pro⁵⁰² in subdomain b2 are involved in the binding of the maltooligosaccharide part of the acarviosyl-maltooligosaccharides, even though Lys⁴⁹³ and Pro⁵⁰² have little contribution to decreasing K_m values for all the substrates (Table 3). In a previous study from our group (16), the role of Ser⁴⁹⁷ (in subdomain b2) in governing the affinity at subsite +4 was demonstrated through site-directed mutagenesis. In agreement with the previous study, the structures of complexes with acarviosyl-maltooligosaccharides revealed the interaction of Ser⁴⁹⁷ with O5 and O6 of ring E through hydrogen bonds. The glucose residues of the maltooligosaccharide part were primarily observed to make contact with remote subsites through van der Waals interactions. The aliphatic side chains of Leu²⁴⁰ and Val⁵⁰¹ significantly interacted with rings E and F/G, respectively. Therefore, the mutant enzyme L240A had a substantially reduced affinity at subsite +4. In contrast, the kinetic characteristics of V501A are in conflict with its structural attribute. Structural analysis suggested that Val⁵⁰¹ likely contributes to the affinity at subsites +5/+6; however, the mutant V501A exhibited reduced affinity at subsite +4. This contradiction could be explained by the effect of the Val⁵⁰¹ mutation on Ser⁴⁹⁷. As shown in our previous report (16), the mobility of the side chain of Ser⁴⁹⁷ is high, resulting in its observation as a dual conformation of the AC4 complex structure. The side chain of Val⁵⁰¹ is located close to the hydroxy group of Ser⁴⁹⁷, and the mutation (Val⁵⁰¹ → Ala) may increase the mobility of the hydroxy group of Ser⁴⁹⁷; such increased mobility is likely to diminish the interaction of the hydroxy group with the substrate, leading to lowered affinity at subsite +4. Furthermore, V501R showed the same substrate specificity as V501A, indicating that an arginine residue at the Val⁵⁰¹ position, which is observed in other plant α-glucosidases, is not enough to increase affinity at subsite +4. It is possible that Val⁵⁰¹ of SBG is one of the key residues to achieve its enormous specificity for long-chain substrates as compared with other plant enzymes.

The higher specificity of SBG for longer substrates, which is responsible for the lower dissociation constant of substrates with high DP from the enzyme, appears to be rationalized by the conformation of rings D–H. Of the rings A–H bound to SBG, rings A–C at the non-reducing terminus are extended and the energy gained by binding with the extended substrate contributes to decreasing the activation energy of the hydrolysis reaction. In contrast, rings D–H remain in the stable conformation, as also observed in cycloamyloses and the helical structures of V-amylose (33). SBG is unlikely to obtain the binding energy required for lowering the activation energy from the binding of rings D–H. The binding of substrates in a stable conformation appears to be advantageous compared with a strained conformation insofar as the decrease in dissociation constant is concerned, providing additional energy is unnecessary. As mentioned above, the higher specificity for longer substrates is responsible for the lower dissociation constant of the substrates with high DP from the enzyme. The subsite structure suitable for native substrate conformation is likely to result in lower K_m for the substrates with high DP without the extra energy. Moreover, SBG is likely to ingeniously use the self-stabilizing property of amylose and soluble starch to form stable ES complexes with these long-chain substrates.

The high specificity of SBG for amylose and soluble starch is also attributable to the band-flip, which is found between rings F and G in structures of AC7 and AC8 complexes. The band-flip observed in the helical structures of the larger cycloamyloses and V-amylose has been generally accepted to be responsible for the relieving strain induced in the macrocycle (34). A likely hypothesis is that AC8 forms the band-flip to alleviate the strain caused, and the structure of SBG is fit to flip. However, the subsite structure of SBG has also been considered to provoke the band-flip; thereby, relieving the strain in long-chain helical substrates leading to the formation of a stable ES complex. In the latter case, the subsite structure likely allows SBG to handle α-1,6-branched structures in the substrate at the trans-oriented point. In the structures of complexes with acarviosyl-maltooligosaccharides, the hydroxy groups of C6 atoms of the rings A–F are close to the enzyme surface, and α-1,6-branched structures are unlikely to be accommodated there. However, the trans-oriented bond results in the O6 atoms of rings G and H facing outward, which would allow the accommodation of α-1,6-branched structures. The extremely higher specificity of SBG for soluble starch (branched substrate) than Glc₁₈ (linear substrate) may be attributable to this mode of substrate binding.

The current study has highlighted the utility of acarviosyl-maltooligosaccharides and provided structural insights into the mechanism that governs the high specificity of SBG for substrates with high DP. The acarviosyl-maltooligosaccharides proved to be useful as inhibitors of SBG. Moreover, kinetic analysis has demonstrated that the binding of the series of acarviosyl inhibitors represents the binding mode of the substrates. Analyses of the structure of the acarviosyl-maltooligosaccharide complexes revealed that subsites remote from the active-site pocket of the enzyme complement the native structure of amylose, which adopts a helical conformation through intramolecular hydrogen bonds. This mode of binding likely leads to lower K_m and higher specificity for longer substrates. The study of acarviosyl-maltooligosaccharides is likely to prove useful for the study of other α-glycosidases as well. For example, glucoamylase shows notable specificity for longer maltooligosaccharides, but the crystal structure of the enzyme has been solved with acarbose (35). The use of acarviosyl-maltooligosaccharides is likely to clarify the mode of substrate binding at subsites far from the active site of glucoamylase.