J. Biochem. 112, 127-131 (1992)

Evidence for a Single Catalytic Site of Honeybee a-Glucosidase I by Chemical Modification with Diethylpyrocarbonate Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060 Received for publication, January 8, 1992

Honeybee a-glucosidase I was inactivated with diethylpyrocarbonate (DEPC). The inactivation followed pseudo-first-order kinetics. The rate of the loss of activity was decreased by the addition of a substrate, maltose. Since there was no spectral change in the tyrosine absorption region, it was recognized that DEPC did not react with this residue. The a-glucosidase had one free sulfhydryl group, which was not involved in the catalytic reaction, and was not modified by DEPC. On the other hand, the specific reaction of DEPC with a histidyl residue was spectrophotometrically confirmed by an increase in absorption near 240 nm, and the activity of the inactivated enzyme was restored by hydroxylamine. The modification rate of one histidyl residue by DEPC was almost equal to the rate of the activity loss. These results indicate that there is one histidyl residue at or near the catalytic site, and that honeybee a-glucosidase I has a single active site.

Honeybees have at least three kinds of a-glucosidases. The two of them, a-glucosidase I and II, were purified as homogeneous proteins (1-3). In the previous papers (1, 4), we reported that a-glucosidase I, a monomeric protein (Mr 9.8 X104), showed Michaelis-Menten type reaction and two different types of allosteric cooperativity, depending on the kind of substrates. The rates of aglycone release from PNPG, phenyl a-glucoside, sucrose and maltose (the reducing terminal glucose for maltose) were accelerated with an increase in substrate concentration (negative kinetic cooperativity). Turanose and amylose (DP = 13) were hydrolyzed with a positive kinetic cooperativity, giving sigmoidal plots of the initial velocity against the substrate concentration. In addition, the hydrolysis of maltooligosaccharides (malto-triose to -octaose), kojibiose, and phenyl a-maltoside, gave hyperbolic MichaelisMenten type reaction curves. Few examples are known of monomeric enzymes that show such unique kinetics. If a monomeric enzyme has more than two catalytic sites which have a cooperative interaction, unusual reactivity may be exhibited (5). Therefore, it is of fundamental interest to elucidate whether honeybee a-glucosidase I has a single active site or two separate active sites. We have attempted to examine the active site of the enzyme by means of chemical modification. Treatment with DEPC, a specific reagent for histidyl residue (6), inactivated the enzyme. The inactivation was found to be caused by the modification of a single histidyl residue located at or near the catalytic site. This suggests that honeybee a-glucosidase I catalyzes the reaction at a single active site and that the atypical kinetic behaviors may not be due to plural Abbreviations: DEPC, diethylpyrocarbonate; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); EDTA, ethylenediaminetetraacetic acid; IAA, iodoacetamide; MNT, 2-methoxy-5-nitrotropone; NEM, iV-ethylmaleimide; PCMB, p-chloromercuribenzoate; PNPG, p-nitrophenyl ff-glucoside; TNBS, trinitrobenienesulfonate; TNM, tetranitrometh-

Vol. 112, No. 1, 1992

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active sites. The present paper concerns with an evidence, obtained by chemical modification, for a single catalytic site of honeybee a-glucosidase I. MATERIALS AND METHODS Chemicals—The sources of chemicals used in this work are as follows: PNPG, maltose (SP grade), DTNB, IAA, PCMB, and NEM were purchased from Nacalai Tesque Chemical; maltotriose, hydroxylamine hydrochloride, and TNBS, from Wako Pure Chemical; maltotetraose and maltopentaose, from Nihon Shokuhin Kogyo; DEPC, from Kanto Chemical; TNM, from Maruwaka Kagaku Kogyo; MNT, from Sankyo. Maltose was purified by repeated recrystallization to remove a possible impurity. The concentration of DEPC was determined by the method of Melchior and Fahrney (7) before use. Enzyme and Assay—Honeybee a-glucosidase I, homogeneous in disc and SDS-gel electrophoresis, was prepared by the method described in the previous paper (1). The enzyme concentration was determined from the values of A}*, at 280 nm of 13.4 and the molecular weight of 9.8 X10* (2). The enzyme reaction was carried out at 35*C in 0.05 M sodium acetate buffer (pH 5.0). The amounts of glucose and p-nitrophenol liberated from a substrate were measured by the same methods as in the previous paper (2). Treatment of the Enzyme with DEPC—DEPC was freshly diluted with ice-cold ethanol for £ach experiment. The enzyme was incubated with 0-6.0 mM DEPC in 0.1 M potassium phosphate buffer (pH 5.5) at 25'C. The final concentration of ethanol in the reaction mixture was less than 1.2%, and ethanol at this concentration did not affect the enzyme stability. The DEPC treatment was terminated by 500-fold dilution with ice-cold 0.05 M sodium acetate buffer (pH 5.8) containing 0.05% Triton-X 100. The residual activity was measured in terms of the amounts of p-nitrophenol and glucose liberated from 0.2% PNPG.

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Atsuo Kimura, Hirokazu Matsui, Mamoru Honma, and Seiya Chiba

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A. Kimura et al.

RESULTS

Inactivation of Honeybee a-Glucosidase I by DEPC— Figure 1A shows the time course of the loss of honeybee ar-glucosidase I activity during incubation with DEPC. Inactivation followed pseudo-first-order kinetics, and increased with an increase in the concentration of DEPC. The pseudo-first-order rate constant (^,bi) was determined from the slope of each curve in Fig. 1 A. As shown in Fig. IB, the Abbs value is directly proportional to the DEPC concentration. From the slope in Fig. IB, the second-order rate constant for the inactivation was estimated to be 1.08 M"' • s~\ The rate of inactivation was apparently reduced by the presence of maltose, which was a good substrate (Fig. 1C). This implies that DEPC reacts with amino acid residue (s) located at or near the active site of the enzyme. However, there is a possibility that a decrease of the affinity of the enzyme for substrate brings about the apparent reduction of the enzyme activity. If the affinity is changed by modification, an alteration in the 2C value may be observed. Figure 2 shows the Lineweaver-Burk plots for hydrolysis of maltooligosaccharides by the native enzyme and the partially modified enzyme having 45% of the original activity. The K^ value for each substrate was not affected by the inactivation of DEPC, and only the Vmai value was decreased; the Km values for maltose, maltotriose, -tetraose, and -pentaose were 0.85, 0.62, 2.0, and 8.0 mM, respectively. Determination of the Amino Acid Residue Modified by DEPC—DEPC is generally regarded as a specific reagent for histidyl residue in protein. However, reaction with tyrosine residue, sulfhydryl group, or amino group has also been reported (6). The O-carbethoxytyrosyl residue formed through the modification with DEPC can be easily detected by the change in the absorbance at 278 nm (9). The difference spectrum between the native and the DEPC-treated enzymes is shown in Fig. 3. There is no change in the absorption of 270 to 280 nm, indicating that tyrosyl residue is not involved in the modification. The increase of the absorbance near 240 nm may be due to the formation of N-carbethoxyhistidyl residue by the reaction of histidyl residue in the protein with DEPC. As listed in Table I, several specific reagents, IAA, PCMB, and NEM for sulfhydryl group, MNT and TNBS for amino group, and TNM for tyrosyl residue, had no or little effect on the activity of the a-glucosidase. When the native ar-glucosidase was treated with DTNB in the absence of denaturant, 0.83 mol of free sulfhydryl group per 1 mol of the

enzyme was modified. No inactivation was caused by the modification. This suggests that the ar-glucosidase has one sulfhydryl group which is not involved in the enzyme reaction. As shown in Table II, both the native and DEPC-treated a-glucosidase have one free sulfhydryl group when measured in 8 M urea, indicating that DEPC did not modify this sulfhydryl group in the enzyme. It has been reported that hydroxylamine removes the carbethoxy group from iV-carbethoxyhistidyl residue to bring about recovery of the activity of the inactivated enzyme (7, 20). The 30% active enzyme preparation was incubated with 1 M hydroxylamine at pH 7.0 and 35"C. As shown in Fig. 4, the enzyme activity was restored completely after 15 h incubation, indicating that the inactivation by DEPC is reversible. Determination of Number of Histidyl Residues—The number of modified histidyl residues was measured in terms of the increase in absorbance at 240 nm, reflecting the formation of iV-carbethoxyhistidine (e240 = 3.2xl0 3 ) (11). Figure 5 A shows the relationship between the number of blocked histidyl residues (mol/mol of protein) and the residual activity during DEPC treatment. The enzyme was completely inactivated at 10 rnin incubation, and two residues were totally modified. As shown in Fig. 5B, the 8emilogarithmic plots of residual histidine content do not give a continuous straight line. This nonlinearity of the plots indicates that the two histidyl residues have different reactivities. It was assumed that there are two types of histidines, referred to as "fast" and "slow" reacting. The line for the loss of fast-reacting histidine was obtained by subtracting the value extrapolated for the slow-reacting one from the value for the loss of total histidyl residues. The rate constant (kobs) for the modification of the fastreacting histidine, calculated from the slope, was estimated to be 6.67 X 10~3 a"1, and that for slow-reacting histidine, 1.92 X 10~s s~\ calculated from the linear portion of the curve obtained experimentally. The curve for the loss of enzyme activity is shown in the same graph, giving a straight line, and the kobi value was estimated to be 6.29 X 10~3 a"1. The rate for modification of the fast-reacting residue is considered to be almost the same as that for the loss of enzyme activity, because the fet* values for the individual rates are nearly equal. The findings indicate that there is one histidyl residue at or near an active site, and that there is a single catalytic site in a molecule of the enzyme. DISCUSSION It has been reported that DEPC is a specific reagent for histidyl residue, though some side reactions (modifications of tyrosyl residue, amino and sulfhydryl groups) occur (6). Because the enzyme activity was little affected by treatment with reagents having relatively high specificity for these moieties (Table I), however, such a residue or group seems not to be located at the active site of honeybee a-glucosidase I. The lack of change in the absorption in the 270 to 280 nm region between the native and modified enzymes also confirms that DEPC did not attack the tyrosyl residue. The absorption near 240 nm, a characteristic of iV-carbethoxyimidazole derivative formation in' protein (6), of the DEPC-treated enzyme increased (Fig. 3), and the modified enzyme was reactivated with hydroxylamine J. Biochem.

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Measurement of Free Sulfhydryl Group—Total free sulfhydryl group of native and DEPC-treated enzymes were spectrophotometrically titrated with 0.4 mM 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) in 0.1 M sodium phosphate buffer (pH 7.5) containing 8 M urea and 10 mM EDTA at 25'C. The reaction was monitored by the measurement of absorption at 412 nm, using a value of e — 1.36xlO 4 M~ 1 -cnr 1 for the released 2-nitro-5-mercaptobenzoate (8). The native a-glucosidase was treated with 2.8 mM DTNB in 0.1 M sodium phosphate buffer (pH 7.5) containing 10 mM EDTA at 25*C for 40 min in the absence of urea. The residual activity and the number of sulfhydryl groups modified by DTNB were measured by the methods described above.

Single Catalytic Site of Honeybee a-Glucosidase I

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5 Reaction time (min)

Reaction time (min) Fig. 2. Ldneweaver-Burk plots for hydrolysis of maltooligosaccharides by native and DEPC-treated honeybee a-glncosidase I. The partially inactivated enzyme having 45% of the original activity was prepared by the same method as described in Fig. 1A (treatment with 1.2 mM DEPC for 10 min). The reaction mixture containing 0.5 ml of substrate solution, 0.25 ml of 0.2 M sodium acetate buffer (pH 5.0) and 0.25 ml of enzyme solution (0.15 //g for DEPC-treated and 0.23 MS for native

Evidence for a single catalytic site of honeybee alpha-glucosidase I by chemical modification with diethylpyrocarbonate.

Honeybee alpha-glucosidase I was inactivated with diethylpyrocarbonate (DEPC). The inactivation followed pseudo-first-order kinetics. The rate of the ...
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