Identification of Catalytic Residues of Ca2+-independent 1,2-{alpha}-D-Mannosidase from Aspergillus saitoi by Site-directed Mutagenesis*

Yota Tatara, Byung Rho Lee, Takashi Yoshida {ddagger}, Koji Takahashi § and Eiji Ichishima 

From the Laboratory of Molecular Enzymology, Graduate School of Engineering, Soka University, Hachioji, Tokyo, 192-8577, Japan, {ddagger}Department of Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, 036-8561, Japan, and §Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183-8509, Japan

Received for publication, March 14, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The roles of six conserved active carboxylic acids in the catalytic mechanism of Aspergillus saitoi 1,2-{alpha}-D-mannosidase were studied by site-directed mutagenesis and kinetic analyses. We estimate that Glu-124 is a catalytic residue based on the drastic decrease of kcat values of the E124Q and E124D mutant enzyme. Glu-124 may work as an acid catalyst, since the pH dependence of its mutants affected the basic limb. D269N and E411Q were catalytically inactive, while D269E and E411D showed considerable activity. This indicated that the negative charges at these points are essential for the enzymatic activity and that none of these residues can be a base catalyst in the normal sense. Km values of E273D, E414D, and E474D mutants were greatly increased to 17–31-fold wild type enzyme, and the kcat values were decreased, suggesting that each of them is a binding site of the substrate. Ca2+, essential for the mammalian and yeast enzymes, is not required for the enzymatic activity of A. saitoi 1,2-{alpha}-D-mannosidase. EDTA inhibits the Ca2+-free 1,2-{alpha}-D-mannosidase as a competitive inhibitor, not as a chelator. We deduce that the Glu-124 residue of A. saitoi 1,2-{alpha}-D-mannosidase is directly involved in the catalytic mechanism as an acid catalyst, whereas no usual catalytic base is directly involved. Ca2+ is not essential for the activity. The catalytic mechanism of 1,2-{alpha}-D-mannosidase may deviate from that typical glycosyl hydrolase.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In Enzyme Nomenclature Recommendations (1), three types of {alpha}-mannosidases, namely {alpha}-D-mannoside mannohydrolase (EC 3.2.1.24 [EC] ), 1,2-{alpha}-mannosyl-oligosaccharide {alpha}-D-mannohydrolase (EC 3.2.1.113 [EC] ), and 1,3-(1, 6)-mannosyl-oligosaccharide {alpha}-D-mannohydrolase (EC 3.2.1.114 [EC] ), are given as systematic names (2, 3). Aspergillus saitoi 1,2-{alpha}-D-mannosidase specifically cleaves the {alpha}-1,2-mannosidic linkage in the high mannose type oligosaccharides of glycoproteins.

Glycosyl hydrolases are classified as retaining and inverting (46). The inverting reaction occurs via a single displacement mechanism with inversion of anomeric configuration. The reaction usually involves two carboxylic acids. These residues are located ~9 Å apart on average. One acts as a general acid catalyst, donating a proton to the glycosidic oxygen of the scissile bond. The other acts as a base catalyst, activating water for nucleophilic attack at the anomeric carbon. The retaining reaction proceeds via a double displacement mechanism with retention of anomeric configuration. The nucleophilic carboxylic acid residue attacks the glycosyl oxygen. A covalent glycosyl enzyme intermediate is formed and hydrolyzed with general acid-base catalytic assistance. In the retaining enzymes, the two carboxylic acid residues are ~5.5 Å apart.

Recently the three-dimensional structures of 1,2-{alpha}-D-mannosidase from Penicillium citrinum were determined by Lobsanov et al. (7). The three-dimensional structures complexed with an inhibitor kifunensin and 1-deoxymannojirimycin demonstrated that both inhibitors bind to the protein at the bottom of the cavity in an unusual 1C4 conformation (4C1 is energetically more stable than 1C4). The inhibitor binding did not undergo major conformational changes. Three carboxylic acids (Glu-122, Asp-267, and Glu-409) were potentially involved in the catalytic mechanism. These correspond to Glu-124, Asp-269, and Glu-411 in the A. saitoi enzyme. The four other highly conserved carboxylic residues were also shown (the equivalent A. saitoi numbering is in parentheses): Glu-271 (Glu-273), Glu-412 (Glu-414), Glu-472 (Glu-474), and Glu-502 (Glu-504). These four residues seemed to be too distant from the substrate to be directly involved in catalytic action. They either are buried at the bottom of the active site or interact with atoms on the inhibitor far from the anomeric C-1. A molecule of Ca2+ is located at the bottom of the active site cavity. Glu-271 (Glu-273), Glu-409 (Glu-411), Glu-412 (Glu-414), and Glu-472 (Glu-474) are hydrogen-bonded to the water molecules that coordinate the Ca2+. The distance of the Ca2+ and an inhibitor/substrate suggests that they can directly interact with each other. Moreover, one of the candidates for catalytic residues, Glu-409 (Glu-411), coordinates the Ca2+ via a water molecule. The Ca2+ is essential for the enzyme activity of the mammalian and yeast 1,2-{alpha}-mannosidases (814). These suggest that Ca2+ may be directly involved in the catalytic reaction of 1,2-{alpha}-D-mannosidases. The amino acid sequence of A. saitoi 1,2-{alpha}-D-mannosidase (15) is 70% identical with that of P. citrinum enzyme (16), and their substrate specificity to a high mannose type oligosaccharide is the same (2, 3, 17). Structures of the active center are believed to be conserved among A. saitoi and P. citrinum enzymes because of their high homology. Although we previously demonstrated the probable roles of these acidic residues (18), their catalytic residues have not yet been determined experimentally.

In this study, we performed site-directed mutagenesis to determine the functional role of catalytic residues in the 1,2-{alpha}-D-mannosidase from A. saitoi. Ca2+, potentially involved in the catalysis, was also analyzed with atomic absorption spectrophotometry. To probe the roles of the three active site carboxylic acids, Glu-124, Asp-269, and Glu-411, in greater detail, we generated the alanine mutants of these residues and assayed these enzymes. Rescue of activity by external anion was expected to provide valuable insight into the identity of the general base catalyst as well as confirmation of the identity of the general acid catalyst (1924).


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The nucleotide sequence of the 1,2-{alpha}-D-mannosidase gene (D49827 [GenBank] ), msdS, was described earlier (15). Aspergillus oryzae niaD300 and a high level expression vector plasmid pNAN8142 were used for transformation experiments (25). Man-{alpha}1,2-Man-OMe1 was purchased from Sigma.

Site-directed Mutagenesis—Site-directed mutagenesis of the 1,2-{alpha}-D-mannosidase gene (msdS) was performed by the method of Kunkel et al. (26). The following oligonucleotides were used as mutagenic primers: E124A, 5'-AGCCTCTTCGcGACCACCATC-3' (msdS position, 361–381); E411A, 5'-TGCGCCCGGcAGTGATTGAgAGCTTCTACT-3' (1222–1252). The E124Q, E124D, D269N, D269E, E273Q, E273D, E411Q, E411D, E414Q, E414D, E474Q, E474D, and E504Q mutants were constructed by inserting the NotI fragment of the mutated genes in pGAM1 (18) into a NotI restriction site in pNAN8142. The plasmid pNAN-AM1 containing wild type or mutated msdS was transfected into the A. oryzae niaD300 strain. The transformation of A. oryzae and the expression of the mutated 1,2-{alpha}-D-mannosidase genes were carried out according to the procedure described previously (25). Purification of recombinant enzymes expressed in A. oryzae cells was also described earlier (25). A pH gradient was substituted for the NaCl gradient in the cation-exchange chromatography. The recombinant enzymes were separated by SDS-PAGE gel as described by Laemmli (27). The proteins were detected by immunoblotting using anti-A. saitoi 1,2-{alpha}-D-mannosidase antibody (raised in rabbit) and peroxidase-conjugated anti-rabbit IgG secondary antibody.

Enzyme Assay—The purified wild type and mutant enzymes were assayed for 1,2-{alpha}-D-mannosidase activity with Man-{alpha}1,2-Man-OMe as a substrate. Mannose from Man-{alpha}1,2-Man-OMe released by the enzymic reaction was stained by the Somogyi-Nelson (28, 29) method. One katal of 1,2-{alpha}-D-mannosidase activity was defined as the amount of enzyme required to liberate 1 mol of mannose from Man-{alpha}1,2-Man-OMe per second at 30 °C and pH 5.0.

The initial rates of 1,2-{alpha}-D-mannosidase activity were determined for a Man-{alpha}1,2-Man-OMe substrate at 30 °C and pH 5.0 in 50 mM acetate buffer using 8 substrate-concentrations ranging from 0.5x to 2x Km. Values of the catalytic coefficient (kcat) and Michaelis constant (Km) values were determined by fitting initial rates as a function of substrate concentration to the direct linear plot of Cornish-Bowden.

Chemical Rescue Methodology—Activities of E124A, D269N, and E411A 1,2-{alpha}-D-mannosidase mutants catalyzing Man6GlcNAc2-PA to Man5GlcNAc2-PA in the presence of external nucleophilic anion at pH 5.0 were analyzed by HPLC analysis. Ten pmol of Man6GlcNAc2-PA (Takara Shuzo Co., Kyoto) with or without 3 M sodium azide was added to the enzyme solution. After 72 h at 30 °C, the reaction mixture was stopped by heating to 100 °C for 3 min and analyzed on HPLC using TSKgel Amide-80 column (4.6 x 250 mm, Tosoh Corp., Tokyo). The solvent and elution conditions were described by Kondo et al. (30).

Ca2+ Binding Treatment—The purified recombinant A. saitoi 1,2-{alpha}-D-mannosidase was used as a Ca2+-free wild type enzyme. NaCl was added to the purified enzyme solution in the final concentration of 1 M. The solution was dialyzed against 10 mM sodium acetate buffer, pH 5.0, containing 50 µM CaCl2 for 16 h at 4 °C and then dialyzed against the same buffer without CaCl2. To analyze the content of Ca2+ binding to the enzyme, the solution was applied to atomic absorption spectrophotometry (Shimadzu model AA-660 (P/N206 –1000-02) apparatus).

Thermal Stability Studies—The stability of the Ca2+-free and Ca2+-bound 1,2-{alpha}-D-mannosidase against thermal-induced unfolding was studied. The unfolding transition curves were obtained by a circular dichroism measurement done in a Jasco J-720 spectropolarimeter. The protein solution was dialyzed against 50 mM formate-KOH buffer, pH 4.0, then diluted in the same buffer to adjust the A280 to 0.2– 0.3. Path length of the optical cuvette was 2 and 10 mm for the measurement of the far and near UV regions, respectively. The fraction of protein in the unfolding state (fU) was determined using the equation fU = ({theta}T {theta}N)/{theta}D{theta}N), where {theta}T is the ellipticity at temperature T, and {theta}N and {theta}D represent the ellipticity for the native and denatured protein, respectively.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Specific Activities of Mutant and Wild Type Enzyme—Seven carboxylic acid residues to be studied were constructed in the cloned A. saitoi 1,2-{alpha}-D-mannosidase gene (msdS) by site-directed mutagenesis. Recombinant enzymes were purified homogeneously on SDS-PAGE gels (Fig. 1A) and identified as A. saitoi 1,2-{alpha}-D-mannosidase by Western blot analysis (Fig. 1B). Purified forms of all recombinant enzymes were assayed by the Somogyi-Nelson method (28, 29). No activity was detected under standard assay conditions for any of the mutant enzymes except E504Q mutant, and the specific activity could only be determined after extended incubation, typically at 60 –90 min (Table I). From Table I, it appears obvious that the mutations generated resulted in enzymes with significantly reduced ability to hydrolyze Man-{alpha}1,2-Man-OMe, as their specific activity is at least 103-fold less than that of the wild type enzyme. The specific activities of D269N and E411Q mutants in particular were not detected completely. The results of several experiments suggest that there is no significant contamination by wild type 1,2-{alpha}-D-mannosidase in the low activity of mutant enzymes (D269N and E411Q).



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FIG. 1.
SDS-PAGE (A) and Western blot analysis (B) of the A. saitoi 1,2-{alpha}-D-mannosidase and mutant enzymes expressed in A. oryzae cells. Proteins were detected using anti-A. saitoi 1,2-{alpha}-D-mannosidase antibody as described under ``Experimental Procedures.'' Positions of the mutated amino acid residues are indicated above the lanes.

 

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TABLE I
Specific activities of purified wild type and mutant 1,2-{alpha}-D-mannosidases

 

Kinetic Analysis—Kinetic characterization of E124Q, E124D, D269E, E273D, E411D, E414D, and E474D mutants and wild type 1,2-{alpha}-D-mannosidase was performed at pH 5.0 using Man-{alpha}1,2-Man-OMe as a substrate. Results of these kinetic experiments are shown in Table II. Kinetic parameters for D269N and E411Q mutants could not be measured, since the reaction rates were below the detectable limit. The kcat value for E124Q mutant was 0.0078 s1, almost 0.3% that of the wild type enzyme. E124D mutant enzyme also decreased the kcat value (0.9% of wild type enzyme). The Km values of E124Q and E124D mutant enzymes were essentially unchanged. Compared with wild type 1,2-{alpha}-D-mannosidase, the D269E and E411D mutant enzymes showed 17- and 28-fold increases in Km values, respectively, whereas the kcat values were slightly lower than that of wild type enzyme. The Km values of E273D, E414D, and E474D mutant enzymes were greatly increased to 23–31-fold that of wild type enzyme, and the kcat values were decreased (1–15% of wild type enzyme).


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TABLE II
Kinetic parameters of wild type and mutant 1,2-{alpha}-D-mannosidases S.E in kinetic parameters: Km (±2.6-10.4 %) and kcat (±1.8-4.8 %).

 

pH-Activity Profiles of E124A and E124D Mutant 1,2-{alpha}-D-Mannosidase—Fig. 2 shows the pH dependence of the reaction rate for Man-{alpha}1,2-Man-OMe hydrolysis for wild type 1,2-{alpha}-D-mannosidase and E124A and E124D mutant enzymes. The optimum pH of E124D and E124A mutant enzymes shifted from 5.0 to 4.0 and 4.5, respectively. The activity of E124A mutant enzyme was found to be lower than that of wild type enzyme over the whole pH range studied, but the effect was more pronounced for the basic limb.



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FIG. 2.
pH-activity profiles of wild type 1,2-{alpha}-D-mannosidase ({circ}), E124D (x) and E124A ({diamond}) mutant enzymes represented as relative activities. The absolute activity value of the E124A mutant at pH 5.0 corresponds to about 0.015% of the wild type enzyme activity.

 

Effects of an External Nucleophile on Alanine Mutant Enzymes—Mutation (Glu or Asp to Ala) at the general base catalyst creates a cavity in the active site that can accommodate a small external nucleophile. Sodium salts of azide at 3 M were used as external nucleophiles in the enzyme-catalyzed hydrolysis of Man6GlcNAc2-PA sugar chain. The reaction mixture was analyzed by HPLC (Fig. 3). The activity of wild type 1,2-{alpha}-D-mannosidase was inhibited by a high concentration of azide. The E124A, D269N, and E411A mutant enzymes were inactive in the absence of azide, as a peak corresponding to a Man5GlcNAc2-PA was not detected. In the presence of azide, these mutant enzymes could not be reactivated.



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FIG. 3.
Effects of an external nucleophile on catalytic residue mutants of A. saitoi 1,2-{alpha}-D-mannosidase using Man6GlcNAc2-PA as a substrate. A mixture (20 µl) of 10 pmol Man6GlcNAc2-PA, 3 M sodium azide, and a mutant or the wild type enzyme was incubated at pH 5.0 (50 mM acetate buffer) and 30 °C for 72 h. A, 0.25 µM wild type enzyme. B, 9.7 µM E124A mutant enzyme. C, 5.9 µM D269N mutant enzyme. D, 10.5 µM E411A mutant enzyme.

 

Atomic Absorption Spectrophotometric Analysis and Kinetic Analysis of Wild Type 1,2-{alpha}-D-Mannosidase—Concentrations of Ca2+ were determined by atomic absorption spectrophotometric analysis. The purified recombinant wild type 1,2-{alpha}-D-mannosidase almost completely did not contain Ca2+ (data not shown). Other divalent metal cations including Mg2+, Mn2+, Co2+, Cu2+, and Zn2+ were also not detected. After Ca2+ treatment (see "Experimental Procedures"), Ca2+ content was determined as 0.9 mol/mol of wild type 1,2-{alpha}-mannosidase. Kinetic parameters of Ca2+-free and Ca2+ binding 1,2-{alpha}-D-mannosidase were determined (Table III); the kcat and Km values were little affected by the Ca2+ binding.


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TABLE III
Kinetic parameters of wild type and Ca2+ binding 1,2-{alpha}-D-mannosidase

 

EDTA Inhibition—EDTA inhibited the Ca2+-free wild type 1,2-{alpha}-D-mannosidase. The inhibition was investigated by fixed concentrations of EDTA in the assay buffer and by varying the concentrations of substrate. A Lineweaver-Burk plot showed a pattern consistent with competitive inhibition (Fig. 4) and gave a Ki of 0.91 mM for EDTA.



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FIG. 4.
Lineweaver-Burk plot of A. saitoi 1,2-{alpha}-D-Mannosidase in the presence of EDTA. The Ca2+-free wild type 1,2-{alpha}-D-mannosidase was assayed as described under ``Experimental Procedures'' at the indicated concentrations of EDTA. •, none; {triangleup}, 0.5 mM; {diamond}, 1 mM; {circ}, 2 mM; x, 5 mM.

 

Ca2+ Binding Properties of Mutant 1,2-{alpha}-D-Mannosidases— Ca2+ concentration of the mutant 1,2-{alpha}-D-mannosidase was determined by atomic absorption spectrophotometry after Ca2+ binding treatment as described under "Experimental Procedures." As shown in Table IV, the Ca2+-free wild type enzyme bound 0.9 mol of calcium/mol of protein, ~1 mol/mol. E124D, E411D, and E504Q mutants also contained 0.8, 1.1, and 0.9 mol of calcium/mol of protein, respectively. E124Q, D269E, D269N, E474Q, and E474D contained less calcium than 1 mol of calcium/mol of protein. E411Q, E273Q, E273D, E414Q, and E414D mutants demonstrated an almost complete loss of binding of Ca2+.


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TABLE IV
Ca2+ binding properties of wild type and mutant 1,2-{alpha}-D-mannosidases after Ca2+ -treatment determined by atomic absorption spectrophotometry

 

Effect of Ca2+ Binding on 1,2-{alpha}-D-Mannosidase Thermal Stability—The spectra of Ca2+-free and Ca2+-bound 1,2-{alpha}-D-mannosidase at far and near UV region were consistent with each other (data not shown). The Ca2+ binding did not undergo a large conformational change. Thermally induced loss of secondary and tertiary structure was monitored at 222 and 291 nm, respectively (Fig. 5). Tm, the temperature at which half of the molecules are unfolded, for Ca2+-free 1,2-{alpha}-D-mannosidase was 56 and 58 °C at 222 and 291 nm, respectively. The Ca2+ binding contributed to the thermal stability increasing Tm to 58 and 62 °C at 222 and 291 nm, respectively.



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FIG. 5.
Thermal transition curves of Ca2+-free ({circ}) and Ca2+-bound 1,2-{alpha}-D-mannosidase ({diamond}) at pH 4.0. Unfolding was monitored by far UV (222 nm) and near UV (291 nm). The mean residue ellipticity was normalized as described under ``Experimental Procedures.''

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Identification of Catalytic Amino Acid Residues—Drastic reductions in the values of the catalytic coefficient (kcat) were observed in the E124Q and E124D mutant enzymes (Table II). This may result from significant impairment of the chemical steps in the catalytic reaction. The Km value was essentially unchanged from wild type enzyme. It is, thus, clear that Glu-124 participates in glucosidic bond hydrolysis. E124D shifted the optimum pH to be more acidic (Fig. 2). This result is shown by the lower pKa of Asp than that of Glu and supports the assumption that Glu-124 is directly involved in the catalytic mechanism. It could not be determined whether the Glu-124 residue is a carboxylate group (COO) or carboxyl group (COOH). The pH dependence of E124A affected the basic limb, which was generally assumed to be due to the general acid catalyst. An external nucleophile sodium azide did not reactivate the E124A mutant enzyme (Fig. 3). Thus the Glu-124 may act as an acid catalyst. The mechanism of the all carbohydrate hydrolases usually involves a pair of carboxylic acid residues. In P. citrinum 1,2-{alpha}-D-mannosidase, two of the three residues (Glu-124, Asp-269, and Glu-411 in A. saitoi enzyme) are believed to be potentially catalytic residues (7). Glu-124 was suggested to act as an acid catalyst as discussed above. The most likely candidates for the other catalytic residues are Asp-269 and Glu-411. However, D269E and E411D did not show a considerable decrease in the kcat values (Table II). Recently, a base catalyst of an inverting {beta}-amylase (EC 3.2.1.2 [EC] ) from Bacillus cereus var. mycoides was determined by the chemical rescue methodology for a catalytic site mutant (E367A) by azide (19). In the case of A. saitoi 1,2-{alpha}-D-mannosidase an external nucleophile did not reactivate the D269N and E411A mutant enzymes (Fig. 3). Neither Asp-269 nor Glu-411 residues can be a base catalyst in the normal sense. Although the D269N and E411Q mutant enzymes were catalytically inactive, the D269E and E411D mutant enzymes showed considerable activity. The results indicate that the carboxyl groups at these positions may be required for the 1,2-{alpha}-D-mannosidase activity. Structural and modeling studies on cellobiohydrolase Cel6A (EC 3.2.1.91 [EC] ) from Trichoderma reesei suggest that the catalytic mechanism may not directly involve a catalytic base (31). The catalytic mechanism of 1,2-{alpha}-D-mannosidase deviates from the typical glycosyl hydrolase.

Role of Glu-273, Glu-414, and Glu-474 Residues—E273D, E414D, and E474D mutant enzymes decreased kcat values and increased Km values (Table II). The reduction of the kcat value is assumed to be due to the destabilization of the sugar ring. It has become increasingly clear that ring distortion at the –1 site is crucial in the catalytic mechanism of cellobiohydrolase (32, 33). The catalytic reaction of {alpha}-amylase from Pseudomonas stutzeri involves three acidic residues; two residues act as a proton donor and a proton acceptor. The other residue works to tightly bind the substrate, giving a twisted and deformed conformation of the glucose ring at position –1 (34). Glu-273, Glu-414, and Glu-474 may bind the substrate to change the conformation of the mannose ring into more reactive one. The three-dimensional structure of P. citrinum 1,2-{alpha}-D-mannosidase indicates that Glu-271, Glu-412, and Glu-472 residues (Glu-273, Glu-414, and Glu-474 in A. saitoi enzyme) in the active site are located at the bottom of the cavity and are not directly involved in the catalytic mechanism. It is demonstrated that these residues are hydrogen-bonded to the Ca2+ via water molecules, and the Ca2+ is involved in the stability of a substrate. But the recombinant wild type A. saitoi 1,2-{alpha}-D-mannosidase contained no Ca2+ and other divalent metal cations. Despite the Ca2+ having no effect on the 1,2-{alpha}-mannosidase activity (Table III), its binding sites were conserved in A. saitoi 1,2-{alpha}-D-mannosidase. This also indicates that the drastic decrease of the activities of E273D, E414D, and E474D mutants (Table IV) did not parallel the loss of the Ca2+ binding ability. These residues are involved in the substrate binding sites.

Ca2+ Binding—It was reported earlier that the recombinant A. saitoi 1,2-{alpha}-D-mannosidase has a molecule of Ca2+ (25). In this study, the recombinant wild type A. saitoi 1,2-{alpha}-D-mannosidase contained no Ca2+. The purified wild type enzyme did not bind Ca2+ with an addition of CaCl2 (data not shown). We found that the Ca2+-free enzyme bound 1 mol of Ca2+ per 1 mol of protein after the addition of a high concentration of NaCl. It was also demonstrated that the Ca2+ binding site is conserved (Table IV). 1,2-{alpha}-Mannosidase generally requires a divalent metal cation for the activity (13, 14, 3537). In the case of A. saitoi 1,2-{alpha}-D-mannosidase, the Ca2+ had no effect on either the kcat or the Km value (Table III). Thus, it is clear that Ca2+ is not essential for the activity of A. saitoi 1,2-{alpha}-D-mannosidase, although the Ca2+ binding site is conserved. EDTA was shown to behave as a competitive inhibitor of A. saitoi 1,2-{alpha}-D-mannosidase (Fig. 4); this indicates that the enzyme contains no divalent metal cation. 1-Deoxymannojirimycin inhibits 1,2-{alpha}-mannosidase as a substrate analog. However, EDTA is not a substrate analog. Tris is a competitive inhibitor for 1,2-{alpha}-mannosidase from rabbit liver (14). Tris and other buffers containing primary hydroxyl groups substantially decreased its activity (38, 39). Carboxyl groups of EDTA may have an affinity for the active site of A. saitoi 1,2-{alpha}-D-mannosidase. A molecule of Ca2+ contributed to thermal stability (Fig. 5). The role of Ca2+ for A. saitoi 1,2-{alpha}-D-mannosidase is to protect the enzyme against thermal denaturation, and it was observed to have the same role in Saccharomyces cerevisiae-processing 1,2-{alpha}-mannosidase (12). Although Ca2+ is essential for S. cerevisiae enzyme activity (12, 37), A. saitoi 1,2-{alpha}-D-mannosidase did not require Ca2+ for the activity (Table III). This is the first report demonstrating that 1,2-{alpha}-D-mannosidase requires no divalent metal cation for its activity.


    FOOTNOTES
 
* 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. Back

To whom correspondence should be addressed. Fax: 81-426-91-9312; E-mail: ichisima{at}t.soka.ac.jp.

1 The abbreviations used are: Man, mannose; Man-{alpha}1,2-Man-OMe, methyl 2-O-{alpha}-D-mannopyranosyl {alpha}-D-mannopyranoside or {alpha}-D-Man-[1-> 2]-{alpha}-D-Man-1-> OMe; GlcNAc, N-acetylglucosamine; OMe, methyl ester; PA, pyridylaminated. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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