A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline, Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate*

Backgroud: A pure and stable transition-state analogue for lysozyme has not been reported thus far. Results: We synthesized 4-O-β-tri-N-acetyl-chitotriosyl moranoline (3), which inhibited strongly the lysozyme reaction. Conclusion: Compound 3 was found to be a novel and stable transition-state analogue for lysozyme. Significance: The crystal structure of lysozyme in a complex with 3 supports the covalent glycosyl-enzyme intermediate in the catalytic reaction. 4-O-β-Di-N-acetylchitobiosyl moranoline (2) and 4-O-β-tri-N-acetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)4, and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with Ki values of 2.01 × 10−5 and 1.84 × 10−6 m, respectively. From ITC analysis, the binding of 3 was found to be driven by favorable enthalpy change (ΔHr°), which is similar to those obtained for 2 and (GlcNAc)4. However, the entropy loss (−TΔSr°) for the binding of 3 was smaller than those of 2 and (GlcNAc)4. Thus the binding of 3 was found to be more favorable than those of the others. Judging from the Kd value of 3 (760 nm), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWL in a complex with 3 showed that compound 3 binds to subsites −4 to −1 and the moranoline moiety adopts an undistorted 4C1 chair conformation almost overlapping with the −1 sugar covalently bound to Asp-52 of HEWL (Vocadlo, Davies, G. J., Laine, R., and Withers, S. G. (2001) Nature 412, 835–838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction.

Lysozymes (EC 3.2.1.17) are glycosidases acting in the innate-immune system of most animals (1). The common feature of lysozymes is to exert antibacterial activity by cleaving the ␤-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine (GlcNAc) 5 in peptidoglycan, a major bacterial cell-wall component (2). Hen egg white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by x-ray crystallography, revealing that the enzyme is divided into two domains by a deep substratebinding cleft containing the catalytic site (3). The catalytic importance of the conserved residues, Glu-35 and Asp-52, was also confirmed by the crystal structure of the enzyme-ligand complex (4) and site-directed mutagenesis (5). These studies on the structure and function of HEWL offer the strongest support available to date for the theory that enzymes may catalyze reactions by binding substrates in the geometry of the transition state more strongly than in that of the ground state.
HEWL has six subsites for sugar residue binding, termed Ϫ4 to ϩ2 (formerly A, B, C, D, E, and F (6)). The enzyme cleave a glycosidic linkage between the sugar residues at subsites Ϫ1 and ϩ1 with the aid of the proton-donating action of Glu-35 as a general acid. The substrate analog tri-N-acetylchitotriose, (GlcNAc) 3 , binds predominantly to subsites Ϫ4, Ϫ3, and Ϫ2 of HEWL, keeping away from subsite Ϫ1 due to the unfavorable positive free energy of the sugar residue binding at that subsite (7). This situation might have been an obstacle to understanding the catalytic mechanism of the enzyme. In the classical explanation, the lysozyme-catalyzed reaction was recognized to take place via a carbenium ion intermediate stabilized by the carboxylate of Asp-52 as a conjugate base. The intermediate state adopts a half-chair conformation with C1 carbon display-ing sp 2 hybridization. Thus, the binding of a pyranose ring to subsite Ϫ1 is likely accompanied by distortion of the pyranose ring from its normal chair conformation toward the half-chair conformation (8,9). Experimental evidence for the distortion of the Ϫ1 sugar was obtained by binding experiments using an unsaturated derivative from cell-wall tetrasaccharide (8) and a ␦-lactone derived from (GlcNAc) 4 (10). The affinity of the ␦-lactone derivative to HEWL was much higher than that of the unsaturated derivative, suggesting that the ␦-lactone derivative is a transition-state analogue for HEWL. However, the ␦-lactone derivative used is unstable and always in a mixture with the gluconic acid. Thus, the data obtained with the lactone derivative might not be accurate. It is highly desirable to obtain a pure and stable transition-state analogue for HEWL to more deeply understand the enzyme mechanism.
We recently reported a synthetic method for easily obtaining the conjugated ␦-lactone, 4-O-␤-tri-N-acetylchitotoriosyl-2-acetamido-2,3-dideoxydidehydroglucono-␦-lactone (5) derived from tetra-N-acetylchitotetraose (GlcNAc) 4 by chemical modification of the reducing end residue of the tetrasaccharide (11). Compound 5 might be a candidate for the stable transition-state analogue for HEWL. Meanwhile, Vocadlo et al. (12) reported the crystal structure of HEWL covalently bound to C1 carbon of the Ϫ1 sugar, which exhibits a chair conformation with C1 carbon in sp 3 hybridization. Because 1-deoxynojirimycin (moranoline) acting as the paradigms of glycosidase inhibitors is known to bind subsite Ϫ1 of the corresponding enzymes (13)(14)(15), chitin oligosaccharides, (GlcNAc) n , modified with moranoline at their reducing end might be another candidate for the stable transition-state analogue for HEWL.
Analytical Methods-HPLC analysis was carried out to determine the amounts of products from the lysozyme-catalyzed transglycosylation reaction using an ANIDIUS column (4.6 ϫ 250 mm, Develosil, Japan) with a JASCO Intelligent system liquid chromatograph and detection by ultraviolet light at 210 nm. The bound material was eluted with 80% CH 3 CN at a flow rate of 1.0 ml/min at 40°C. The HRESI-MS spectra were measured on a JMS-T100LC mass spectrometer. 500-MHz 1 H NMR spectra and 125-MHz 13 C NMR spectra were recorded using a JEOL -500 spectrometer. Chemical shifts were expressed in ppm relative to the methyl resonance of the external standard, sodium 3-(trimethylsilyl)propionate.
Enzymatic Synthesis of Compounds 1, 2, and 3 by HEWL-(GlcNAc) 4 (0.90 g, 1.1 mmol) and moranoline (1.24 g, 7.6 mmol) were dissolved in 50 ml of 50 mM acetate buffer (pH 4.5) containing 50% DMSO, and then HEWL (1.0 g, 100,000 kU) was added to conduct the transglycosylation reaction shown in Scheme 1. The mixture was incubated for 100 h at 50°C. The precipitate was removed by centrifugation (27,600 ϫ g, 20 min) and the supernatant was directly applied to an ion-exchange  column (Dowex-50W H ϩ form, 3.0 ϫ 18 cm) equilibrated with H 2 O. After washing the column with H 2 O, the adsorbed portion was eluted with 0.5 M NH 4 OH. The adsorbed portion was concentrated and dissolved in 10 ml of H 2 O and then loaded onto a Toyopearl HW-40S column (5.5 ϫ 60 cm). The column was developed with the 0.1 M NaCl at a flow rate of 80 ml/h and a fraction size of 7 ml/tube. The chromatogram identified four distinct peaks, F-1 to F-4 (Fig. 2B). Fractions corresponding to the individual peaks were pooled, dialyzed, concentrated, and lyophilized.
Lysozyme Inhibition Assays-IC 50 was determined by measuring the lysozyme activity in the presence of inhibitors, using a turbidity assay under the following conditions. The reaction mixture (0.15 ml) comprising bacterial cell suspensions of M. lysodeikticus (0.2 mg/ml) in 100 mM phosphate buffer (pH 7.0), and 0 to 1.0 mM inhibitor was preliminarily incubated at 25°C for 5 min. Finally, the HEWL solution (2.5 l, 50 units) in the same buffer was added. The decrease in OD 450 nm of the cell suspension was monitored for 2.5 min using a UV-visual spectrophotometer V-630 (Jasco Co., Tokyo, Japan). IC 50 values for the inhibitors were calculated from Dixon and Webb plots (17). In addition, the modes of inhibition were examined for the individual compounds, 2, 3, and 5, by means of Lineweaver-Burk plots (18), which were also used for calculation of the K i values. The experimental conditions were as follows. The reaction mixture (0.03 ml) comprising 36 -280 M pNP-(GlcNAc) 5 , 100 mM sodium acetate buffer (pH 5.5), and an inhibitor (0 -0.2 mM for 2, 0 -0.02 mM for 3, or 0 -0.2 mM for 5) was preliminarily incubated at 40°C for 5 min. The reaction was performed with 63 units of HEWL. Samples (4 l) were taken at intervals (0, 5, 10, 15, 20, and 25 min) and inactivated by boiling at 100°C for 5 min. The amounts of pNP-GlcNAc and pNP-(GlcNAc) 2 formed by the enzymatic reaction were determined by HPLC using the same system as described above except that the detection was performed by ultraviolet light at 300 nm.
ITC Experiments-The HEWL solution (45 M) in 20 mM phosphate buffer (pH 6.0, 7.0, and 8.0) was degassed and its concentration was determined using absorbance of ultraviolet light at 280 nm. Individual (GlcNAc) n (n ϭ 3 and 4) and the chitooligosaccharide derivatives (1 mM (GlcNAc) 3  Analysis of Calorimetric Data-ITC data were collected automatically using the Microcal Origin version 7.0 software accompanying the iTC200 system. Prior to data fitting, all data were corrected for heat of dilution by subtracting the heat remaining after saturation of binding sites of the enzyme. The magnitude of the heat after the saturation was similar to that obtained for the ligand titration into the buffer alone. Nonlinear least-squares fitting to the experimental data using a single-site binding model was satisfactory, providing reliable values of the stoichiometry (n), equilibrium binding association constant (K a ), and the reaction enthalpy change (⌬H r°) for the lysozymeinhibitor interaction. The value of n was found to be within the range from 0.9 to 1.2 for all interactions. The binding free energy change (⌬G r°) and the entropy change (⌬S r°) were calculated using Equation 1.
⌬G r°ϭ ϪRTlnK a ϭ ⌬H r°Ϫ T⌬S r°( Eq. 1) Crystallization and Data Collection-Crystallization conditions for HEWL in complex with compounds 2 or 3 were screened using the sparse-matrix sampling method, by sitting drop vapor diffusion at 20°C. Under optimized crystallization conditions, 1 l of protein solution (17 mg/ml in water) containing 1.2 mM 2 or 3 was mixed with 1 l of reservoir solution containing 0.2 M magnesium chloride hexahydrate, 0.1 M Bis-Tris (pH 6.5), and 25% polyethylene glycol 3350 (PEG 3350). Crystals grew within 3 days to a size of up to 0.3 ϫ 0.6 ϫ 0.3 mm 3 . For data collection, the crystals were transferred into a cryoprotectant solution containing 0.2 M magnesium chloride hexahydrate, 0.1 M BisTris (pH 6.5), 25% PEG 3350, and 30% glycerol and then flash-cooled in a nitrogen stream at 95 K. The diffraction data were collected at beamline BL-17A of Photon Factory (Ibaraki, Japan), using an ADSC Q315 CCD detector, at a cryogenic temperature (95 K). The data were integrated and scaled with HKL2000 (19). The processing statistics are summarized in Table 1.
Structural Determination and Refinement-The structures of HEWL in complex with compounds 2 and 3 were solved by the molecular replacement method using the program MOLREP (20), where the structure of a hen egg white lysozyme, PDB code 1BWJ (21), served as a search model. One protein molecule was located in the crystallographic asymmetric unit. The model was improved by several rounds of refinement with REFMAC (22) and manual rebuilding with COOT (23). The structure of the complex with 2 was refined to R work /R free of 0.168/0.181 at a resolution of 1.19 Å. The final model of the complex with 2 contains one protein molecule that includes residues 1-128, compound 2, and 171 water molecules. The structure of the complex with 3 was refined to R work /R free of 0.167/0.174 at a resolution of 1.19 Å. The final model of the complex with 3 contains one protein molecule that includes residues 1-128, compound 3, and 148 water molecules. The stereochemistry of the models was verified using PROCHECK (24). The complex with 2 showed 88.4, 11.6, 0.0, and 0.0% of protein residues in the most favored, additionally allowed, generously allowed, and disallowed regions of the Ramachandran plot, respectively. The complex with 3 showed 89.3, 10.7, 0.0, and 0.0% of protein residues in the most favored, additionally allowed, generously allowed, and disallowed regions of the Ramachandran plot, respectively. Molecular graphics were illustrated with PyMol software (www.pymol.org/). The refinement statistics are summarized in Table 1.
Protein Data Bank Entry-The atomic coordinates and structural factors have been deposited in the Protein Data Bank, under accession codes 4HPI (complex with 2) and 4HP0 (complex with 3).

Enzymatic Synthesis of Compounds 1, 2, and 3 by HEWL-
We have already reported the regioselective synthesis of p-nitrophenyl penta-N-acetylchitopentaoside, pNP-(GlcNAc) 5 , through HEWL-catalyzed transglycosylation from (GlcNAc) 5 as a donor substrate to the C4-position of p-nitrophenyl N-acetylglucosaminide as an acceptor (25). Following the procedure was developed for pNP-(GlcNAc) 5 synthesis, we tried to produce compounds 1, 2, and 3 from (GlcNAc) 4 by the use of HEWL (Scheme 1). A regioselective transglycosylation from the (GlcNAc) 4 donor to the moranoline acceptor successfully took place, producing a series of 1, 2, and 3. The time course of the enzymatic-transglycosylation reaction is shown in Fig. 2A, which was obtained by HPLC determination of the reaction products. The transfer reaction led to the preferential formation of 1 rather than 2 and 3. The yields of 2 and 3 did not increase even if a longer incubation was conducted. The products were easily separated from each other by successive column chromatographies of Dowex-50W and Toyopearl HW-40S. The gel-filtration profile is shown in Fig. 2B. Compounds 1, 2, and 3 were finally obtained in yields of 36.3, 5.9, and 2.7%, respectively, based on the amount of (GlcNAc) 4 added. The structures of these compounds were confirmed by 1 H and 13 C NMR analyses in D 2 O solution and also by HRESI-MS analysis, as described under "Experimental Procedures."  MARCH 1, 2013 • VOLUME 288 • NUMBER 9

JOURNAL OF BIOLOGICAL CHEMISTRY 6075
Inhibition Analysis-The inhibitory effects of compounds 1, 2, and 3, 4-O-␤-tri-N-acetylchitotriosyl-2-acetamido-2,3-dideoxydidehydroglucopyranose (4), and 4-O-␤-tri-N-acetylchitotoriosyl-2-acetamido-2,3-dideoxydidehydroglucono-␦-lactone (5) (Scheme 1 and Fig. 1) on the lytic activity of HEWL toward M. lysodeikticus cells were compared with those of (GlcNAc) n (n ϭ 2, 3, and 4), which were used as reference compounds. The results are listed in Table 2. At pH 7.0 the IC 50 value of 3 (0.57 M) was one-fourteenth of that of (GlcNAc) 4 (7.7 M), which is preferentially bound in a nonproductive manner. In fact, at saturation of the enzyme with (GlcNAc) 4 , only 0.11% of the tetrasaccharide demonstrates productive modes of binding (26). Compounds 2 and 5 also acted as inhibitors and the effects were approximately equivalent to that of the reference compound (GlcNAc) 3 . Compound 3 was found to be the most effective inhibitor of lysozyme lysis. Comparison between the data for 3 and (GlcNAc) 4 led to an important finding that the moranoline moiety of 3 is most significantly responsible for the inhibitory action of this compound.
We then tried to determine the inhibition constants of these inhibitors. Although turbidimetry is widely used for lysozyme assays using bacterial cells, this method is less quantitative due to the heterogeneity of the bacterial cell powder and is not appropriate for kinetic measurements. Thus, a kinetic analysis in the presence of the inhibitors was conducted using a well defined low molecular weight substrate, pNP-(GlcNAc) 5 . The use of pNP-(GlcNAc) 5 as the substrate provided fully reliable kinetic data, as shown in Fig. 3. The mode of inhibition of compounds 2, 3, and 5 toward HEWL was found to be competitive based on the Lineweaver-Burk plots. The K i values were graphically determined to be 2.01 ϫ 10 Ϫ5 M for 2, 1.84 ϫ 10 Ϫ6 M for 3, and 3.51 ϫ 10 Ϫ5 M for 5, respectively, by the method of Dixon and Webb (17). The inhibitory activity of 3 was 10-fold higher than those of the others. We also confirmed that the three inhibitors tested were not hydrolyzed by HEWL during the individual inhibition experiments.
ITC Analysis of Binding of Synthetic Inhibitors to HEWL-Interactions of the synthetic inhibitors, 2, 3, and 5, with HEWL were studied by ITC at 25°C and pH 7.0. Fig. 4 shows ITC thermograms and theoretical fits to experimental data for these inhibitors, and the reference compounds (GlcNAc) 3 and (GlcNAc) 4 . Theoretical fits were successfully obtained based on the experimental data using a nonlinear least-squares algorithm by varying the binding affinity constant (K a ), the number of binding sites, i.e. the stoichiometry of the reaction (n), and the enthalpy change of ligand binding (⌬H). For all compounds, all fits yielded n between 0.9 and 1.2 as listed in Table 3, indicating that the binding stoichiometry is 1:1. The binding of lysozyme to (GlcNAc) 3 was confirmed to be enthalpically driven (⌬H r°ϭ Ϫ13.5 kcal/mol), but counterbalanced by an unfavorable entropic contribution (ϪT⌬S r°ϭ 6.6 kcal/mol), resulting in Ϫ6.9 kcal/mol of ⌬G r°, in agreement with the data reported by Garcíia-Hernández et al. (27). A similar ⌬G r°v alue was obtained for 2, in which the reducing end GlcNAc residue of (GlcNAc) 3 is substituted with moranoline moiety. However, a decrease in favorable enthalpy change (from Ϫ13.5 to Ϫ11.0 kcal mol) was observed in 2, and this was compensated for by a decrease in unfavorable entropic contribution (from 6.6 to 4.4 kcal/mol). Although the ⌬H r°v alue for the binding of 3 was similar to those of 2 and (GlcNAc) 4 , the entropy loss (ϪT⌬S r°ϭ 2.6 kcal/mol) was considerably lower in 3, resulting in the highest affinity of 3 (⌬G r°ϭ Ϫ8.4 kcal/mol; K d ϭ 0.76 M) among the inhibitors tested ( Table 3). Substitution of the reducing end residue of (GlcNAc) 4 with moranoline creates a 10-fold improvement in affinity. The affinity (⌬G r°, K d ) for 5 was comparable with those of (GlcNAc) 3 and (GlcNAc) 4 .    A Novel Transition-state Analogue for Lysozyme MARCH 1, 2013 • VOLUME 288 • NUMBER 9 Table 4 lists the values of thermodynamic parameters for 3 obtained at various pH values. The affinity for 3 was found to decrease at a higher pH, 8.0, due to the considerable decrease in favorable enthalpy change.
Crystal Structures of HEWL in Complex with Compounds 2 and 3-HEWLs in complex with 2 and 3 adopted similar conformations to that of unbound HEWL (PDB code 1DPX, Ref. 28), whose structure could be superimposed with a root mean square deviation value of 0.163 and 0.214 Å, respectively, over the corresponding 128 C␣-atoms of the individual complex structures. Fig. 5 shows the simulated annealing omit maps of compounds 2 and 3 bound to HEWL. In both cases, clear electron density for the individual sugar units was found at glyconbinding site (negatively numbered subsites). The moranoline moiety was located at subsite Ϫ1, whereas the remaining GlcNAc residues bound to subsites Ϫ2, Ϫ3, and Ϫ4. It is well known that (GlcNAc) 3 and (GlcNAc) 4 bind to subsites Ϫ2, Ϫ3, and Ϫ4, keeping away from subsite Ϫ1 because of the unfavorable positive binding free energy at that subsite. Thus, the structures shown in Fig. 5 suggest that the moranoline moiety has a strong affinity toward subsite Ϫ1. This is the most important requirement for designing an effective inhibitor for lysozyme. However, more careful comparison between the two complex structures indicated that the moranoline moiety of 3 is more closely located to the Asp-52 side chain than that of 2.
The amino acid residues responsible for the binding of compounds 2 and 3 can be identified from Fig. 6. In the complex structure with 2 (Fig. 6A), the nitrogen atom of the moranoline moiety significantly interacts with the carboxylate of Asp-52. The hydrogen bonding network mediated by a water molecule is also formed between the nitrogen atom of the moranoline moiety, the Glu-35 side chain, the C6-OH of the moranoline moiety, and the Val-109 main chain -NH. In the other side of subsite Ϫ1, the C2-OH of the moranoline moiety appears to interact with the side chains of Asp-48 and Asn-59 through a water molecule. In contrast to the strong interaction of the Ϫ2 sugar of the HEWL-(GlcNAc) 3 complex (PDB code 1HEW, Ref. 29), the GlcNAc residue at subsite Ϫ2 was found to interact only with the Ala-107 main chain C ϭ O. As for the Ϫ3 sugar, in addition to the face-to-face stacking interaction with the indole side chain of Trp-62, several hydrogen bonds are formed with Asp-101 and Asn-103.
Compound 3 appears to more strongly interact with HEWL (Fig. 6B). In fact, an additional interaction of the moranoline moiety was found between the Asn-46 side chain and the C2-OH of the moranoline moiety. The Ϫ2 GlcNAc residue forms several interactions with Asn-59, Trp-62, and Trp-63, in addition to that with Ala-107. The Asp-48 side chain also has a water-mediated interaction with the C6-OH of the Ϫ2 sugar. As in the case of HEWL in complex with 2, the Ϫ3 sugar has a face-to-face stacking interaction with the indole side chain of Trp-62 and a hydrogen bond with the side chain of Asp-101, but the interaction with the Asn-103 side chain is missing. Two hydrogen bonds are formed between the Asp-101 side chain and the Ϫ4 sugar. The great difference in the binding affinities between 2 and 3 may be due to the slight shift in the location of 3 from that of 2 in the substrate-binding cleft of HEWL (Fig. 5).

DISCUSSION
The moranoline molecule, which has been recognized to interact with the catalytic base (or the nucleophile) of the targets, glycoside hydrolases (13), may mimic the transition state of enzymatic hydrolysis. To mimic the transition state of the lysozyme-catalyzed reaction, we designed and tried to produce 4-O-␤-N-acetylglucosaminyl moranoline (1), 4-O-␤-di-N-acetylchitobiosyl moranoline (2), and 4-O-␤-tri-N-acetylchitotriosyl moranoline (3), in which the reducing end GlcNAc residues of (GlcNAc) 2 , (GlcNAc) 3 , and (GlcNAc) 4 are substituted with the moranoline, respectively. HEWL successfully catalyzed a regioselective transglycosylation from the (GlcNAc) 4 donor to the moranoline acceptor, producing compounds 1, 2, and 3. However, the enzyme produced 1 in preference to 2 and 3 in the entire course of the reaction (Fig. 2A). Compound 3 was obtained in only a low yield. A major fraction of (GlcNAc) 4 prefers to bind nonproductively to the lysozyme-binding cleft occupying subsites Ϫ4, Ϫ3, and Ϫ2, with the additional GlcNAc extending beyond the substrate-binding cleft at the nonreducing end (26). Thus, the second (GlcNAc) 4 molecule binds to the vacant subsites, Ϫ1, ϩ1, and ϩ2, resulting in hydrolysis into the nonreducing end GlcNAc and the remaining (GlcNAc) 3 (30). After releasing the (GlcNAc) 3 fragment, the nonreducing end GlcNAc is transferred to the moranoline molecule, producing a higher amount of 1. Compounds 2 and 3 might have been produced from the productive HEWL-(GlcNAc) 4 complexes, in which (GlcNAc) 4 occupies subsites Ϫ2 to ϩ2 and subsites Ϫ3 to ϩ1, respectively, via the bond cleavage and the moranoline transfer reaction. Fractions of the productive HEWL-(GlcNAc) 4 complex are much lower than that of the nonproductive complex occupying subsites Ϫ4, Ϫ3, and Ϫ2 (26), resulting in the lower yields of 2 and 3.
The inhibitory activities of 1 and 2 were comparable with those of (GlcNAc) 2 and (GlcNAc) 3 , respectively (Table 2), indicating that the moranoline moiety itself does not enhance the binding ability to lysozyme. It is well known that the moranoline molecule alone strongly binds to subsite Ϫ1 of glucosidases and inhibits their enzymatic reaction (13)(14)(15). However, it appears that the moranoline does not bind to the lysozyme by itself. The ␦-lactone derivative (5) containing sp 2 hybridization and adopting a stable half-chair conformation also exhibited a similar inhibitory activity to that of (GlcNAc) 3 . The inhibitory activity of 4 was even lower than those of (GlcNAc) 3

and 5.
However, only compound 3 was found to strongly inhibit the lysozyme activity. The stable binding of (GlcNAc) 3 to subsites Ϫ4, Ϫ3, and Ϫ2 (sites A, B, and C) of HEWL and a suitable location of the moranoline moiety might have cooperatively enhanced the binding ability of 3, explaining the highest inhibitory activity of the compound. Kinetic determination afforded K i values of 2.01 ϫ 10 Ϫ5 , 1.84 ϫ 10 Ϫ6 , and 3.51 ϫ 10 Ϫ5 M, and by ITC the binding free energy changes were determined to be Ϫ6.6, Ϫ8.4, and Ϫ6.9 kcal/mol, for 2, 3, and 5, respectively ( Table 3). The experimental data for the IC 50 values, K i values, and binding free energy changes were consistent with each other. Judging from the K d value of 3 (0.76 M), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. The pH dependence of ⌬G r°f or 3 (Table 4) might be derived from the change in the protonation state of the moranoline moiety, of which the pK a value was reported to be 6.3-6.7 (13,31). The protonation state of Glu-35 (pK a ϭ 6.7, Ref. 32), which strongly interacts with the moranoline moiety of 3 (Fig. 6B), might have partly affected the binding ability of the compound.
X-ray crystallography of the structures of HEWL in complex with 2 and 3 revealed significant differences in binding mode  between the two inhibitors. As shown in Fig. 7A, the location of 3 is slightly shifted to the Asp-52 side as compared with that of 2. This situation resulted in the interaction of C2-OH of the moranoline moiety with the Asn-46 side chain. In addition, the (GlcNAc) 3 moiety of 3 bound to HEWL almost completely overlapped with (GlcNAc) 3 bound to HEWL (PDB code 1HEW; Fig. 7B), whereas the (GlcNAc) 2 moiety of 2 did not overlap with the Ϫ2 and Ϫ3 sugars of (GlcNAc) 3 bound to HEWL. The significant shift of the binding mode of 2 from that of 3 might have eliminated several interactions found in the HEWL complex with 3, resulting in the lower binding affinity and the lower inhibitory activity of 2.
Thermodynamic parameters (Table 3) obtained by ITC experiments afforded further information on the binding mechanism. As seen from the values of ⌬G r°, compound 3 (Ϫ8.4 kcal/mol) more strongly interacts with HEWL than the other ligands (Ϫ6.6 to Ϫ7.0 kcal/mol). The interactions were found to be driven by favorable enthalpy change (⌬H r°) with smaller entropy loss (ϪT⌬S r°) . However, the ⌬H r°v alue for 3 was similar to those for 2 and (GlcNAc) 4 . The stronger interaction of 3 appears to be caused by entropy loss (ϪT⌬S r°ϭ 2.6 kcal/mol) lower than those of 2 and (GlcNAc) 4 (4.4 and 3.7 kcal/mol). In the crystal structures (Fig. 6, A and B), the interaction of HEWL with 3 appears to be more intensive than that with 2. The intramolecular interactions within the lysozyme molecule might have been suppressed upon the binding of 3, resulting in an almost equal contribution of favorable enthalpy changes for (GlcNAc) 4 , 2, and 3 (⌬H r°ϭ Ϫ10.7-11.0 kcal/mol).
As stated in the Introduction, the lysozyme-catalyzed reaction was historically recognized to take place via an oxocarbenium ion intermediate adopting a half-chair conformation with the C1 carbon in sp 2 hybridization (8,9). However, the situation has been changed after the report of a crystal structure of HEWL covalently bound to C1 carbon of the Ϫ1 sugar, which exhibits a chair conformation with the C1 carbon in sp 3 hybridization (12). Nowadays, the catalytic mechanism through the covalently bound intermediate is more widely accepted by enzyme researchers (33)(34)(35). However, the crystal structure capturing the covalent glycosyl-HEWL intermediate was obtained only by use of the inactive mutant HEWL(E35Q) and 2-acetamido-2-deoxy-␤-D-glucopyranosyl-(134)-2-deoxy-2-fluoro-␤-D-glucopyranosyl fluoride (NAG2FGlcF). A covalent intermediate of HEWL was also obtained by using a mutant enzyme, in which Asp-52 is mutated to glutamic acid (D52E). In this case, the catalytic mechanism was altered after the D52E mutation, hence the structure is unlikely to reflect the trait of the wild-type HEWL (36). We here presented evidence in support of the covalent glycosyl-enzyme intermediate in the reaction catalyzed by wild-type HEWL. Compound 5 with sp 2 hybridization and compound 3 with sp 3 hybridization are excellent probes for examining the catalytic mechanism for the lysozyme. As seen from Tables 2 and 3, compound 3 binds to HEWL more strongly than compound 5. The moranoline moiety of 3 with sp 3 hybridization appears to mimic the transition state of the lysozyme-catalyzed reaction. Fig. 8 shows a superimposition of compound 3 bound to HEWL and NAG2FGlcF covalently bound to HEWL(E35Q) (12). The Ϫ1 sugars of both ligands are in a chair conformation without distortion, and almost overlap at the ring C4, C5, C6, and N(O) positions. The partial overlap of the moranoline moiety of the bound compound 3 with the Ϫ1 sugar of the covalently bound NAG2FGlcF appears to demonstrate the covalent glycosyl-HEWL intermediate. Evidence of a covalent intermediate using a wild type enzyme was obtained for an invertebrate lysozyme from Tapes japonica (37), which is highly similar to HEWL in their three-dimensional structure and in the catalytic mechanism. Taken together, we concluded that compound 3 is a stable and pure transition-state analogue with sufficient inhibitory activity for wild-type HEWL, providing additional evidence for a covalent glycosyl intermediate.