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J. Biol. Chem., Vol. 279, Issue 8, 7287-7295, February 20, 2004

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Structural and Enzymatic Analysis of Soybean -Amylase Mutants with Increased pH Optimum^*

Akira Hirata, Motoyasu Adachi, Atsushi Sekine, You-Na Kang, Shigeru Utsumi, and Bunzo Mikami

From the Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

Received for publication, August 25, 2003 , and in revised form, November 17, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Comparison of the architecture around the active site of soybean -amylase and Bacillus cereus -amylase showed that the hydrogen bond networks (Glu³⁸⁰- (Lys²⁹⁵-Met⁵¹) and Glu³⁸⁰-Asn³⁴⁰-Glu¹⁷⁸) in soybean -amylase around the base catalytic residue, Glu³⁸⁰, seem to contribute to the lower pH optimum of soybean -amylase. To convert the pH optimum of soybean -amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean -amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The K_m values of the mutants were almost the same as that of soybean -amylase except in the case of M51T, while the V_max values of all mutants were low compared with that of soybean -amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu³⁸⁰ and Asn³⁴⁰ was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu³⁸⁰ and Lys²⁹⁵ was also disrupted. These results indicated that the reduced pK_a value of Glu³⁸⁰ is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean -amylase compared with that of the bacterial -amylase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
-Amylase (-1,4-glucan maltohydrolase, EC 3.2.1.2 [EC] ) catalyzes the liberation of -anomeric maltose from the non-reducing ends of starch and glycogen. -Amylase has been classified into family 14 of 91 glycoside hydrolase families (last updated October 6, 2003) according to the method of Henrissat et al. (1, 2) and is distributed in higher plants and bacteria (3, 4). The cDNAs from higher plants, including soybean (5, 6), barley (7), rye (8), Arabidopsis thaliana (9), and sweet potato (10), and from bacteria, including Bacillus cereus (11), Bacillus polymyxa (12, 13), Clostridium thermosulfurogenes (14), and Bacillus megaterium DSM319 (15), have been cloned and sequenced. The three-dimensional structures of soybean (16, 17), barley (18), sweet potato (19), and B. cereus (20, 21) -amylase have already been determined. All of the previously determined structures of -amylase exhibit a well conserved (/)₈-barrel fold in the core domain and an active center in the cleft of the barrel. The structural analysis of the soybean -amylase (SBA)¹-maltose complex indicated that Glu¹⁸⁶ and Glu³⁸⁰ play important roles in the enzymatic reaction as a general acid and a base catalyst, respectively (16). This finding is supported by the results of site-directed mutagenesis (22) and affinity labeling (23). In the case of B. cereus -amylase (BCB), Glu¹⁷² and Glu³⁶⁷ act as the general acid and base catalyst, respectively, corresponding to Glu¹⁸⁶ and Glu³⁸⁰ in soybean -amylase (20, 21). It has been reported that only bacterial -amylase has the ability to digest raw starches (24-27), an activity that has been ascribed to its C-terminal raw starch-binding domain (11, 20, 28). Except for the raw starch digestive ability, properties of higher plant -amylases differ from those of bacterial enzymes in terms of optimum pH, specific activity, isoelectric points, and the number of sulfhydryl/disulfide groups (29). The optimum pH of higher plant -amylases is around 5.4, whereas that of bacterial enzymes is 6.7. Fig. 1 shows the comparison of structures near Glu³⁸⁰ in both SBA and BCB. The hydrogen bond between the side chains of Glu³⁸⁰ and Lys²⁹⁵ (Glu³⁶⁷ and Lys²⁸⁷ in BCB), which is conserved in both enzymes, seems to be important for -amylase catalysis. In SBA, the hydrogen bond networks (Glu³⁸⁰-(Lys²⁹⁵-Met⁵¹) and Gln⁸⁷-Asp¹⁷⁶- Glu¹⁷⁸-Asn³⁴⁰-Glu³⁸⁰) stabilize the negatively charged form of Glu³⁸⁰, therefore Glu³⁸⁰ in SBA may have a pK_a lower than that of Glu³⁶⁷ in BCB as this type of stabilization was not found in BCB (20). The S of Met⁵¹ is located near N of Lys²⁹⁵ (3.5 Å) and O-2 of Glu³⁸⁰ (3.7 Å), possibly forming a hydrogen bond with N of Lys²⁹⁵. These hydrogen bond networks in SBA are not present in BCB except in the case of the isolated hydrogen bond between Tyr¹⁶⁴ and Thr³²⁸. These five amino acid residues (Met⁵¹, Glu⁸⁷, Asp¹⁷⁶, Glu¹⁷⁸, and Asn³⁴⁰ in SBA) that differ between SBA and BCB are conserved in each of the higher plants and in bacterial -amylase, respectively (16). This difference in architecture around the base catalyst between SBA and BCB is thought to contribute to the difference in the pH optimum of the respective enzymes. To explore the role played by these amino acid residues, we focused on the Met⁵¹, Glu¹⁷⁸, and Asn³⁴⁰ of SBA. These three residues are located near Glu³⁸⁰ of the five residues and are considered as candidates for the control of the pK_a of Glu³⁸⁰.

View larger version (33K): [in this window] [in a new window]   FIG. 1. Stereodiagrams of the structural conformations around the base catalytic residue Glu380 of SBA (yellow) superimposed onto the BCB (cyan) structure. The hydrogen bonds are shown by broken lines. This figure was generated using MOLSCRIPT (54) and Raster3D (55).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Site-directed Mutagenesis—Mutant SBA genes were constructed by a double PCR method. The PCR was performed in a reaction volume totaling 50 µl and containing 2.5 units of TAKARA EX Taq polymerase, 10x buffer, 200 µM dNTP, two synthesized mutagenesis primers at 100 ng/ml, and 5 ng/µl pKSBA (pKK233-2 vector including the SBA gene), which had been constructed previously (6). The following primers, with the desired changes indicated in bold, were used in the mutagenesis procedures (F and R denote the upstream and the downstream primers, respectively): M51T-F, 5'-GGTTACCGTTGATGTGTGGTGGGGG-3'; M51T-R, 5'-CCGTCAACACCTGCAGCTCGAAGCTG-3'; E178Y-F, 5'-GTTGGGCTTGGCCCTGCAGGAGAGC-3'; E178Y-R, 5'-GTAAATGTCTATAATAAGTCCAGAT-3'; N340T-F, 5'-GCCATTTTTGACCTTTACATGTCTTG-3'; N340T-R, 5'-ATGATGTCTAGACAGCATTCTTGCA-3'. The mutagenesis primers were extended using a thermal cycler (model 2400, PerkinElmer Life Sciences) for the PCR (denaturation for 30 s at 95 °C, annealing for 20 s at 55 °C, and elongation for 5 min at 72 °C for 18 cycles). The PCR products were separated by electrophoresis on a 1.2% (w/v) agarose gel, and the products were purified using glass powder (Bio-Rad). The resulting (mutant) 1.5-kilobase pair fragments were blunted by a blunting kit, phosphorylated by T4 polynucleotide kinase at the 5'-segment, and self-ligated using a ligation kit, version 2 (Takara). The constructed mutant vectors were transformed into Escherichia coli strain JM105 using heat shock. The mutant DNA sequences were confirmed by a DNA sequencer.

Preparation of Mutant Proteins—Protein expression was induced by the addition of isopropyl--D-thiogalactopyranoside to reach a final concentration of 1.0 mM in E. coli strain JM105. The purification and activity assay of the mutant SBA were performed by a method described previously (6).

Enzyme Kinetics and pH Dependence of -Amylase Activity—The values of k_cat and K_m for both SBA and the mutants were measured using potato amylopectin as a substrate (30). Concentrations of the substrate were varied from 1/10 to 5 times the K_m value. Calculations of V_max and K_m were performed using the Michaelis-Menten equation with KaleidaGraph Software (Synergy Software). The pH-dependent activities of SBA and the mutants were determined using potato amylopectin at 37 °C in 0.05 M Britton-Robinson buffer at pH 3.0-9.0. The ionic strength of the buffer solution at each pH was adjusted to be 0.2 with NaCl. The apparent pK₁ and pK₂ values were calculated with the KaleidaGraph nonlinear curve-fitting program using Equation 1 (31),

(Eq. 1)

where v and V are specific activity (units/mg) at each pH and pH-independent activity, respectively. [H] is the concentration of hydrogen ions, and K₁ and K₂ are dissociation constants of catalytic groups of the enzyme.

Estimation of Dissociation Constants of the Mutant-Maltose Complexes—The dissociation constants (K_d) of SBA and the mutants respectively complexed with maltose were estimated by the titration of emission spectra based on tryptophan residues (excitation = 295 nm, emission = 330 nm) in 0.1 M acetate buffer at pH 5.4 and 25 °C; observations were made with a Hitachi F-3000 fluorescence spectrometer.

Crystallization and Data Collection—Based on the crystallized condition of the enzyme in a previous report (6), crystallization of wild-type and mutant SBA was performed at 4 °C by the hanging drop vapor diffusion method, i.e. mixing 5 µl of a 24 mg/ml protein solution in 0.1 M sodium acetate buffer (pH 5.4) with 5 µl of reservoir liquid of 48% ammonium sulfate containing 0.1 M sodium acetate buffer, 1 mM EDTA, and 18 mM 2-mercaptoethanol at pH 5.4. Crystals grew for 1 month. The wild-type and mutant crystals used for data collection were 1 x 0.5 x 0.5 mm. These crystals were all trigonal and belonged to P3₁21; the cell dimensions are shown in Table I. The wild-type and mutant crystals were soaked in the mother liquor in the presence of 200 mM maltose for 30 min at 20 °C, and then the samples were mounted in thin walled glass capillaries filled with the mother liquor and sealed with wax. We avoided crystal freezing to prevent unexpected changes, such as a shift in the pH, from occurring. X-ray data of the wild-type crystal was collected at up to 1.86-Å resolution at room temperature using CuK radiation with a Rigaku RAXIS IIC detector coupled to a Rigaku rotating anode generator, and the data were processed with Rigaku software (Rigaku). X-ray data of the respective mutant SBA crystals were collected at up to 1.95-2.1-Å resolution at room temperature using CuK radiation with a Bruker Hi-Star area detector coupled to a MAC Science M18XHF rotating anode generator, and the results were processed with the SADIE and SAINT software packages (Bruker).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Effect of pH on Mutant -Amylase Activity—In the preset study, the three mutants, M51T, E178Y, and N340T, were all shown to have an increased pH optimum as indicated in Fig. 2. The specific activity for the hydrolysis of potato amylopectin at 37 °C in each optimum pH of the respective -amylase mutants is given in Table II. M51T, E178Y, and N340T had decreased specific activities of 11, 43, and 32%, respectively, of the wild-type SBA. The relative pH activity profiles were bell-shaped except in the case of N340T (Fig. 2). Although we did not examine the pH dependence of the V_max and K_m values separately, the relative pH activity profiles appeared to be mainly due to the V_max value; notably the K_m value of the wild type was reported previously to be constant from pH 3.0 to 7.0 (34). Thus, the observed specific activities were fitted to Equation 1, assuming two pK_a values. Based on this curve-fitting, the optimum pH and postulated pK_a values of the catalytic residues were calculated (Table II). The optimum pH values of the three mutants were shifted from pH 5.4 (wild type) to 6.5 (M51T), 6.0 (E178Y), and 6.6 (N340T), all of which are close to that of BCB (pH 6.7). The relative pH activity profiles were explained by the two pK_a values, pK₁ and pK₂, of the assumed basic and acidic catalysts, respectively. These values for the wild type were in agreement with values reported previously for -amylases from soybean (34, 35), sweet potato (36), and barley (37). The pK₁ values of the mutants increased about 0.8-0.9 pH units from that of the wild-type enzyme, while the pK₂ values were almost the same as that of the wild-type enzyme. These results indicated that the mutations affected the environment of the base catalyst and that due to these mutations the changes in the base catalyst were more significant than those in the acid catalyst, resulting in the increased pK₁ characteristic of the mutant enzymes.

View larger version (19K): [in this window] [in a new window]   FIG. 2. pH activity profiles of SBA (), M51T (), E178Y (), N340T (x), and BCB () at 37 °C. The inset shows the fitting curve of SBA and the mutants as calculated using Equation 1.

View larger version (113K): [in this window] [in a new window]   FIG. 3. Stereoviews of the structural conformation of SBA and the mutants complexed, respectively, with maltose. A, SBA (pH 5.4); B, M51T (pH 5.4); C, E178Y (pH 5.4); D, E178Y (pH 7.1); and E, N340T (pH 5.4). The electron density map (green, 2Fo - Fc, contoured at 1.0 ) and the residues around the base catalyst and maltose are illustrated. The hydrogen bonds are indicated by broken lines (black). The mutated residues and the disordered conformations of maltose and the side chain of Glu380 in M51T are cyan. The disrupted hydrogen bonds in each mutants are indicated by broken lines (pink). This figure was generated using BOBSCRIPT (56) and Raster3D (55).

The Structure of M51T-Maltose Complex—Fig. 3B shows the 2F_o - F_c map and the residues around Glu³⁸⁰ of M51T with the bound maltose determined at pH 5.4. In the structure of M51T, the substitution of Thr⁵¹ created a space that had been occupied by S and C in the original Met⁵¹. Instead of being occupied by the side chain of Met⁵¹, this space was occupied by one water molecule, which was hydrogen-bonded to O-2 of Glu³⁸⁰ (2.8 Å) and N of Lys²⁹⁵ (3.2 Å). The loss of the side chain of Met⁵¹ resulted in the disruption of the hydrogen bond between N of Lys²⁹⁵ and O-2 of Glu³⁸⁰ (from 2.9 to 3.8 Å), which may have allowed for the splitting of the side chain of Glu³⁸⁰ into two alternate positions. Both of the positions were shifted by 0.6 and 2.1 Å from that of the Glu³⁸⁰ O-2 in the wild-type SBA. The disposition of Glu³⁸⁰ O-2 also disrupted the hydrogen bond between O-2 of Glu³⁸⁰ and N-2 of Asn³⁴⁰. The altered side-chain position of Glu³⁸⁰ was so close to O-1 of the distorted glucose at subsite -1 that it prevented the binding of -anomeric glucose and the localization of a catalytic water molecule, which would be in accord with the low activity of M51T.

In contrast to the electron density map of the wild-type enzyme, that of maltose in M51T was interpreted to show a mixture of three maltose molecules bound at subsites -2 to -1 (a distorted glucose and an -⁴C₁ glucose at subsite -1), at subsites +1 to +2, and at subsites +2 to +3. It is assumed that a maltose molecule can bind at subsites +1 to +2 only when a distorted glucose residue binds at subsite -1. If an -anomeric glucose residue binds at subsite -1, the second maltose will bind at subsites +2 to +3 due to a collision between O-1 of the glucose at subsite -1 and O-4 of the glucose at subsite +1. The occupancy refinement of the two alternate positions of maltose coupled with the alternate position of the side chain of Glu³⁸⁰ showed that their respective occupancies were both about 0.5.

The Structure of E178Y-Maltose Complex—Fig. 3C shows the 2F_o - F_c map and the residues around Glu³⁸⁰ of E178Y with the bound maltose determined at pH 5.4. The substituted side chain of Y178 created a novel hydrogen bond with the side chain of Asn³⁴⁰ (O Tyr¹⁷⁸-O-1 Asn³⁴⁰, 2.8 Å). The side chain of Asn³⁴⁰ was found to flip toward O of Tyr¹⁷⁸ by rotating the ² torsion angle about 53°, resulting in the disruption of the hydrogen bond between O-2 of Glu³⁸⁰ and O-1 of Asn³⁴⁰. Two maltose molecules were found, one at subsite -2 to -1 and one at subsite +2 to +3. The anomer of the glucose residue at subsite -1 was clearly identified as having the -configuration, suggesting that the collision of this O-1 with O-4 of the glucose residue at subsite +1 prevented the second maltose from binding at subsite +1. Instead of a glucose residue at subsite +1, there were four water molecules. One water molecule was assigned as the catalytic water molecule, and it was found to be in almost the same position as that of O-1 of the distorted glucose residue at subsite -1 in the wild-type SBA-maltose complex. The alternative position of the -anomeric glucose residue at subsite -1 may be ascribed to the increased pK_a value of Glu³⁸⁰, which was induced by the disruption of the hydrogen bond with Asn³⁴⁰. It is thought that both distorted maltose and normal maltose bind primarily at subsite -2 to -1, depending on the protonated and deprotonated states of Glu³⁸⁰.

To examine this hypothesis, the structure of the E178Y-maltose complex was determined at pH 7.1 as shown in Fig. 3D. The orientations of the side chains of Glu³⁸⁰, Asn³⁴⁰, and Tyr¹⁷⁸ were the same as those found at pH 5.4. In contrast to the results obtained at pH 5.4, both a distorted maltose and a normal -anomeric maltose bind at subsites -2 to -1, and the second maltose binds to subsites +1 to +2 or subsites +2 to +3 as found in the case of M51T-maltose complex at pH 5.4 (Fig. 3B). It is suggested that only the deprotonated form of Glu³⁸⁰ can enable the binding of the distorted maltose to subsites -2 to -1.

The Structure of N340T-Maltose Complex—Fig. 3E shows the 2F_o - F_c map and the residues around Glu³⁸⁰ of N340T with the bound maltose determined at pH 5.4. O-1 of the substituted Thr³⁴⁰ was found to face the side chain of Glu¹⁷⁸, creating a weak hydrogen bond with O-1 of Glu¹⁷⁸ at a distance of 3.4 Å and resulting in disruption of the original hydrogen bond between O-2 of Glu³⁸⁰ and O-1 of Asn³⁴⁰. Two separated maltose molecules were found, one at subsites -2 to -1(-anomer) and one at subsites +2 to +3 as was the case for E178Y at pH 5.4. The distance between O-1 of Glu³⁸⁰ and oxygen of the catalytic water molecule was found to be 2.9 Å, which is slightly longer than that found in E178Y. The temperature factor of this catalytic water molecule was also slightly higher in N340T (35 Ų) than in E178Y (31 Ų). These parameters of the catalytic water molecule may account for the decreased specific activity in these mutants and for the fraction of bound distorted maltose at subsites -2 to -1 around their respective pH optima.

Structural Changes Required for Shifts in pH Optimum—Fig. 4 shows the superimposition of the residues around Glu³⁸⁰ in the mutants and the wild-type SBA onto those of BCB. In the mutant M51T, the substituted side chain of Thr⁵¹ formed a hydrogen bond with the side chain of Gln⁸⁷ (O-1 of Thr⁵¹-O-2 of Gln⁸⁷, 2.8 Å) instead of the disrupted two interactions between S of Met⁵¹ and N of Lys²⁹⁵ and N of Lys²⁹⁵ and O-2 of Glu³⁸⁰. The space was filled with a water molecule that formed a hydrogen bond with O-2 of Glu³⁸⁰ and N of Lys²⁹⁵. These changes led to the instability of the side chain of Glu³⁸⁰, resulting in the two disordered alternate positions. The disruption of the hydrogen bond between O-2 of Glu³⁸⁰ and N-2 of Asn³⁴⁰ and the disordered side chain of Glu³⁸⁰ were in accord with the increased pH optimum and the most decreased activity of the mutant (Tables II and III). The situation in the case of BCB was similar in that two water molecules occupied the vacant space between the substituted Thr⁵¹ and the catalytic base residue. But in the case of BCB, the other water molecule was hydrogen-bonded to O of Thr⁴⁷ (3.2 Å), O-2 of Glu³⁶⁷ (3.2 Å), and N of Lys²⁸⁷ (2.8 Å), indicating that these water-mediated hydrogen bonds stabilized the position of Glu³⁶⁷. The results for M51T suggest that the side chain of Met⁵¹ or the water mediated-hydrogen bonds is important for fixing the position of the base catalyst.

View larger version (39K): [in this window] [in a new window]   FIG. 4. Stereodiagrams of the residues around Glu380 in the mutants and the wild-type SBA superimposed onto those of BCB (yellow and black, SBA; cyan, BCB; green, M51T; blue, E178Y; and violet, N340T). The hydrogen bonds are indicated by broken lines. This figure was generated using MOLSCRIPT (54) and Raster3D (55).

In the structures of E178Y and N340T determined at pH 5.4, the binding mode of maltose was altered from subsites -2 to -1 and +1 to +2 (wild type) to -2 to -1 and +2 to +3, respectively, due to the predominant binding of an -anomeric glucose residue at subsite -1. In every case in which a distorted glucose residue binds at subsite -1, the sugar rings are distorted to form conformations that resemble boat or half-chair conformations. Alteration of the binding mode occurred due to changes in pH, as shown in Fig. 3D, in the case of E178Y, suggesting that the binding of a distorted glucose residue at subsite -1 required the deprotonation of Glu³⁸⁰. The strong nucleophile of the ionized side chain of Glu³⁸⁰ may have enabled the distortion of the sugar ring at subsite -1. The position of O-1 in the distorted glucose residue at subsite -1 was very close to that of a catalytic water molecule found in the binding of -glucose at the subsite, suggesting that the ionized side chain of Glu³⁸⁰ can activate the catalytic water molecule to attack C-1 of the glucose residue, provided that a true substrate with an -configuration binds at subsite -1. Thus, the catalytic activity is thought to be very sensitive to the nucleophilic nature of Glu³⁸⁰ in terms of the distance between O-1 of Glu³⁸⁰ and the catalytic water molecule; this sensitivity may account for the decreased activity of E178Y and N340T relative to that of the wild-type enzyme.

Hydrogen Bond Networks Altering the pH Optimum in Other Glycoside Hydrolases—Alteration of the pH optimum by changing the pK_a of the acid/base catalyst in other members of the glycoside hydrolase family has been observed previously (41-43). In a study of Aspergillus awamori glucoamylase (GA) and Bacillus circulans xylanase (BCX), hydrogen bonds between the acid/base catalyst and residues at the active site were shown to play significant roles in regulation of the pH optimum (41, 43). To provide a general concept to describe the control of the pH optimum of enzymes, we compared the active site of SBA with those of GA and BCX. Fig. 5 shows the conformations of active site residues of SBA-maltose, GA-acarbose, and BCX-2FXb (2-deoxy-2-fluoro-xylobiose), which were arranged such that they had the same substrate orientation; in each case, the two catalytic residues were positioned above and below the substrate, respectively. Like SBA, GA (glycoside hydrolase family 15) is an inverting exoglycosidase (1, 2) that produces -D-glucose by hydrolyzing -1,4- and -1,6-glucosidic linkage from the non-reducing ends of starch and related oligo- and polysaccharides (44, 45). Two carboxyl groups are known to be involved in the catalytic mechanism of GA in which Glu¹⁷⁹ and Glu⁴⁰⁰ are the acid/base catalyst (A. awamori and Aspergillus niger numbering system) and correspond to Glu¹⁸⁶ and Glu³⁸⁰ in SBA, respectively (46-49). Glu⁴⁰⁰ in GA also forms hydrogen bond networks (Tyr⁴⁸-Glu⁴⁰⁰-Ser⁴¹¹ and Glu⁴⁰⁰-Gln⁴⁰¹-Asn³¹⁵ including catalytic water) (Fig. 5B). Frandsen et al. (48) conducted a mutational analysis of Y48W, and suggested that Tyr⁴⁸ is functionally linked to Glu⁴⁰⁰ and is important for maintaining the active site geometry and for the stabilization of an oxocarbonium ion intermediate. The k_cat value of Y48W in GA was reduced 80-100-fold, while K_m was increased 2-3-fold. Y48W had unusually high activity at pH values below 4.0. In contrast, Fang et al. (41) reported that disruption of the hydrogen bond between O of Ser⁴¹¹ and O-1 of Glu⁴⁰⁰ by site-directed mutagenesis of Ser⁴¹¹ to either Ala (S411A) or Cys (S411C) reduced the catalytic efficiencies by only 46 and 26% that of the wild-type enzyme, respectively, but the mutant S411A and S411C increased the pH optima when maltose or maltoheptaose was used as the substrate by 0.8-0.9 pH units; this latter effect was mainly due to the increase in the pK₁ of Glu⁴⁰⁰. Although they did not confirm the loss of the hydrogen bond between Ser⁴¹¹ and Glu⁴⁰⁰ by x-ray crystallographic analysis, the increase in the pK₁ values of S411A or S411C in GA was about the same as that found in SBA mutants (shown in Table II), suggesting that the effect of the disruption of the hydrogen bond between Ser⁴¹¹ and Glu⁴⁰⁰ in S411A or S411C of GA corresponds to that of the disruption of the hydrogen bond between Asn³⁴⁰ and Glu³⁸⁰ in both E178Y and N340T in SBA (Fig. 3, C and E).

View larger version (25K): [in this window] [in a new window]   FIG. 5. Stereoviews of the active site residues (green) and substrate (cyan) in SBA, GA, and BCX. The acid/base and nucleophile catalysts are shown in dark gray. CW refers to the catalytic water molecule. A, the structure of SBA complexed with maltose (Protein Data Bank code 1Q6C). B, the structure of GA complexed with acarbose (Protein Data Bank code 1AGM [PDB] ). C, the structure of BCX complexed with 2FXb [PDB] (Protein Data Bank code 1BVV [PDB] ). The hydrogen bonds are indicated by broken lines. This figure was generated using MOLSCRIPT (54) and Raster3D (55).

In this study, we demonstrated that the pH optimum of SBA mutants could be shifted toward the alkaline region by removing hydrogen bonds with the side chain of Glu³⁸⁰, the catalytic base, possibly as a result of increasing its pK_a. The present study also suggested that the formation or deformation of hydrogen bonds between the catalytic residue and residues near the active site can control the pH optimum of enzymes; such changes in hydrogen bonding can lead to shifts in the pH optimum toward either the acidic or alkaline region.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 1Q6C, 1Q6D, 1Q6E, 1Q6F, and 1Q6G) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a grant for the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-774-38-3763; Fax: 81-774-38-3764; E-mail: mikami{at}kais.kyoto-u.ac.jp.

¹ The abbreviations used are: SBA, soybean -amylase; BCB, Bacillus cereus -amylase; GA, glucoamylase (1,4--D-glucan glucohydrolase; EC 3.2.1.3 [EC] ); BCX, Bacillus circulans xylanase (endo-1,4--xylanase; EC 3.2.1.8 [EC] ); 2FXb [PDB] , 2-deoxy-2-fluoro-xylobiose.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. Yasuo Hata and Hiroaki Kato of the Institute for Chemical Research, Kyoto University, for technical advice regarding the data collected by the Rigaku detector. Computation time was provided by the Super-Computer Laboratory, Institute for Chemical Research, Kyoto University.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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