1 identification of the catalytic residues of bifunctional glycogen

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Identification of the catalytic residues of bifunctional glycogen debranching enzyme

Akifumi Nakayama1#, Keizo Yamamoto2, and Shiro Tabata2*

1Nara Prefectural Hospital, Hiramatsu, Nara City, Nara 631-0846, Japan; 2 Department of Chemistry, Nara Medical University, Kashihara, Nara 634-8521, Japan.�

Running Title : Catalytic residues of Glycogen Debranching Enzyme

* Correspondence to S. Tabata, Department of Chemistry, Nara Medical University, Kashihara,

Nara 634-8521, Japan

# Current address: Nara Prefecture Institute of Public Health, Ohmori-cho, Nara city, Nara

630-8131, Japan

Telephone No.: +81-744-29-8810

Fax: +81-744-29-8810

E-mail: [email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 25, 2001 as Manuscript M102192200 by guest on January 30, 2018

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1The abbreviations used are: GDE, glycogen debranching enzyme; glucosidase,

amylo-1,6-glucosidase; transferase, oligo-1,4->1,4-glucantransferase; TAA, Taka-amylase A;

CGTase, cyclodextrin glycosyltransferase; β-CD, cyclomaltoheptaose ( β-cyclodextrin );

Glc-β-CD, 6-O-α-D-glucosyl cyclomaltoheptaose (6-O-α-D-glucosyl-β-cyclodextrin);

Glc5-β-CD, 6-O-α-maltopentaosyl cyclomaltoheptaose (6-O-α- maltopentaosyl

-β-cyclodextrin); IPTG, isopropyl –β-D-thiogalactopyranoside; SDS-PAGE, sodium dodecyl

sulfate-polyacrylamide gel electrophoresis; β-CD Sepharose 6B, β-cyclodextrin immobilized

Sepharose 6B. The mutations are described with the one-letter code; i.e. D535N is a mutant in

which N in the mutant is substituted for D in the wild-type.

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SUMMARY

Eukaryotic glycogen debranching enzyme (GDE) possesses two different catalytic

activities, oligo-1,4->1,4-glucantransferase /amylo-1,6-glucosidase, on a single polypeptide chain.

To elucidate the structure-function relationship of GDE, the catalytic residues of yeast GDE were

determined by site-directed mutagenesis. Asp-535, Glu-564 and Asp-670 on the N-terminal half,

and Asp-1086 and Asp-1147 on the C-terminal half were chosen by the multiple sequence

alignment or the comparison of hydrophobic cluster architectures among related enzymes. Five

mutant enzymes, D535N, E564Q, D670N, D1086N and D1147N, were constructed. The mutant

enzymes showed the same purification profiles as that of wild-type enzyme on β-CD Sepharose

6B affinity chromatography. All the mutant enzymes possessed either transferase activity or

glucosidase activity. Three mutants, D535N, E564Q and D670N, lost transferase activity but

retained glucosidase activity. On the contrary, D1086N and D1147N lost glucosidase activity but

retained transferase activity. Furthermore, the kinetic parameters of each mutant enzyme

exhibiting either the glucosidase activity or transferase activity did not vary markedly from the

activities exhibited by the wild-type enzyme. These results strongly indicate that the two activities

of GDE, transferase and glucosidase, are independent and located at different sites on the

polypeptide chain.


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INTRODUCTION

Glycogen debranching enzyme (GDE) is a bifunctional enzyme exhibiting both the

transferase (oligo-1,4->1,4-glucantransferase [EC 2.4.1.25]) and glucosidase

(amylo-1,6-glucosidase [EC 3.2.1.33]) activity (1). Both enzymes reside on a single

polypeptide chain (2,3) and each has its own distinct catalytic activity. GDE is a key enzyme in

the carbohydrate metabolism of mammalian cells and yeast. GDE along with phosphorylase

ensures the complete degradation of glycogen and the release of glucose-1-phosphate and glucose.

A maltosyl or maltotriosyl residues from the branched chains of glycogen is first transferred to

the main chain by the transferase to expose the α-1,6-glucosyl stub, that is in turn hydrolyzed by

the glucosidase thus allowing glycogen phosphorylase to degrade the linearized α-(1,4) polymer.

Genetic deficiency of the enzyme in human Type III glycogen storage disease (GSD-III or Cori’s

disease) is characterized by hepatomegaly, hypoglycemia, variable myopathy, and

cardiomyopathy (4,5).

GDE have been purified and characterized from rabbit (2,3) and yeast (6,7). Inhibitors

specifically affecting either the transferase or the glucosidase activity provided evidence that each

of the two activities occurred at distinct catalytic sites (8). Liu et al. (9) showed that the

transferase activity were irreversibly inactivated by carbodiimide in the presence of amines

without affecting the glucosidase activity, and concluded the existence of two distinct active sites,

although the locations were not defined. The amino acid sequence analysis of rabbit GDE


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indicated that N-terminal half may encompass the transferase activity, leaving the glucosidase

activity for the C-terminal half (10).

Several facts pointed to the resemblance of yeast GDE to the mammalian GDEs. The

yeast, human and rabbit GDEs have 1536, 1515 and 1555 amino acid residues, respectively, as

deduced from nucleotide sequences (10-12). The yeast GDE N-terminal half also possesses the

four conserved sequences in the α-amylase family, and the C-terminal half displays about 50 %

identity with the C-terminal half of other mammalian GDEs. Foremost, yeast GDE exhibits the

same transferase and the glucosidase action toward glycogen and branched cyclodextrins as those

of mammalian GDEs (7). Therefore, yeast GDE could serve as a model of the eukaryotic one for

elucidating the structure-function relationship.

Aiming to know the locality of the active sites of both the transferase and glucosidase, we

constructed several mutant yeast GDEs by site-directed mutagenesis. From the analysis of the

enzymatic activities, the amino acid residues involved in the catalysis of the transferase and

glucosidase of yeast GDE were identified.


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EXPERIMENTAL PROCEDURES

Chemicals - Maltopentaose and 6-O-α-D-glucosyl cyclomaltoheptaose (Glc-β-CD) were

purchased from WAKO Chemicals. 6-O-α-Maltopentaosyl cyclomaltoheptaose (Glc5-β-CD) were

prepared as described previously (7). The QuickChange Site-Directed Mutagenesis kit was

purchased from Stratagene Corp and Bicinchoninic acid (BCA) protein assay reagent kit was

from Pierce Chemicals.

Bacterial strain, plasmids and media - Escherichia coli JM105 [thi-1, rpsL, endA, sbcBC,

hsdR4∆ (lac-proAB), [F’ traD36, proAB, laclq Z∆M15] ] was used as a host for the expression of

the wild-type and mutated GDE genes. Plasmid pTrcDBE that express the recombinant GDE

were constructed in a previous study (11). Luria-Bertani (LB) medium containing 50 µg/ml

ampicillin was used for cultivating the E.coli .

Site-Directed Mutagenesis - Plasmids harboring GDE gene with single site mutation were

constructed by using QuickChange Site-Directed Mutagenesis kit. The mutagenesis primers were

synthesized by TaKaRa Syuzo Custom service. DNA sequencing via the dideoxy

chain-terminating method (13) with dsDNA serving as a template confirms the site and type of

mutations occurring on the gene.


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Expression and purification of wild-type and mutant GDEs - The expression and

purification of the wild-type and mutant GDEs were performed as described previously (11).

E.coli JM 105 harboring recombinant plasmid bearing wild-type or mutant GDE gene were

grown in LB medium containing 50µg/ml of ampicillin at 25 oC to an A600 = 0.6 before

isopropyl-β-D-thiogalactopyranoside (IPTG) was added at the final concentration of 0.02 mM.

Induction of the enzymes was carried out for 12 h at 25 oC, and the cells were harvested by

centrifugation at 5,500 x g for 5 min. The collected cell pellet was resuspended in 20 mM

Tris-HCl buffer (pH 7.5) and sonicated. To the supernatant obtained by centrifuging the

sonicated samples at 17,000 x g for 20 min was added ammonium sulfate to a final concentration

of 0.7 M. The crude lysate was then applied to a β-CD Sepharose 6B affinity column

previously equilibrated with 20 mM Tris-HCl buffer, pH 7.5/0.7 M ammonium sulfate. The

column was washed with the same buffer repeatedly before eluting the enzyme with a linear

gradient of ammonium sulfate (0.7-0 M) in 20 mM Tris-HCl buffer (pH 7.5). Homogeneity of the

enzymes was analyzed by sodium dodecyl sulfate-polyacrilamide gel electrophoresis

(SDS-PAGE) and the protein was visualized by staining with Coomassie brilliant blue R-250.

Protein concentration was determined by using the BCA protein assay reagent kit, with bovine

serum albumin as the standard (14).

HPLC Analysis of the sugar products - Transferse and glucosidase activities of the mutant

enzymes were analyzed by HPLC using branched cyclodextrins Glc-β-CD and Glc5-β-CD as a


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substrate, respectively (7). A 10 µl reaction mixture contains 25 mM citrate buffer pH6.5, 3 µg of

purified enzyme and 10 mM Glc-β-CD or Glc5-β-CD substrate. The reaction was carried out at

37 oC for 1 or 5 h, and the enzyme was inactivated by 30 sec of boiling over a water bath. The

reaction mixture was then 5-fold diluted with distilled water, and filtered through ultrafree-MC

(30,000 cut-off filter, Millipore). An aliquot of the filtrate was injected into HPLC (BioLC,

Dionex, Sunnyvale, CA) and allow to adsorb on a CarboPac PA-1 column (250 x 4 mm) for 3

min under eluent of 150 mM NaOH. The mixtures of branched cyclodextrin products were eluted

by increasing linearly the sodium acetate concentration in 150 mM NaOH from 0-33 % for 45

min at the flow rate of 1.0 ml/min. The sugar was detected by a pulsed amperometric detector

(PAD-II) with a gold working electrode and triple-pulsed amperometry.

Enzymatic assays for transferase and glucosidase acitivities - The kinetic parameters for

transferase activity were measured according to the method of Tabata et al. (15) using

maltopentaose as a substrate. The reaction mixture (25 µl) containing varied concentration of

maltopentaose in 40 mM phosphate buffer (pH 6.5) and 5 µl of enzyme solution was incubated at

37 oC for 10 min. The products formed were analyzed by HPLC as described above. Velocity of

the transferase activity at 37 oC, pH 6.5 was expressed as the sum of the products maltose and

maltotriose (nmol /min).

Glucosidase activity was determined by measuring the amount of glucose released from

the substrate Glc-β-CD as described previously (11). The reaction mixture (50 µl) contained 50


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mM citrate buffer (pH 6.5) and appropriate amount of Glc-β-CD. The reaction was initiated by

the addition of 10 µl of enzyme solution and was carried out at 37 oC for 10 min. The enzyme

reaction was terminated by 30 s boiling over a water bath. The released glucose was determined

spectrophotometrically by monitoring the reduction of NADP using hexokinase and glucose

6-phosphate dehydrogenase (16). One unit of activity was defined as the amount of enzyme

catalyzing the release of 1 µmol glucose/min at 37 oC, pH 6.5.

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Analysis of the amino acid sequences for the prediction of amino acids essential for GDE

activity - As shown in Fig. 1, alignment of the amino acid sequences of human muscle (12),

rabbit muscle (10) and yeast (11) GDE revealed the presence of four consensus sequence

commonly found in the α-amylase family or glycosyl hydrolase family 13 (17) on the N-terminal

half. It has been suggested that the consensus sequence II, III and IV in the α-amylase family

contain three completely conserved acidic amino acids (two Asp and one Glu) (18). Jespersen et

al. (19) suggested that the corresponding carboxyl residues in human GDE are involved in the

transferase activity. Therefore, amino acid residues, Asp-535, Glu-564 and Asp-670, which are

located in the consensus sequence II, III and IV of yeast GDE, respectively were chosen as the

target for site-directed mutagenesis to determine their role on the transferase activity.

On the other hand, Liu et al. suggested that C-terminal half might encompass the

glucosidase activity (10). However, the amino acid sequence of yeast GDE (11) showed that its

C-terminal half had no significant homology with other amylolytic enzymes except for the

mammalian GDEs (10, 12). Likewise, the amino acid sequence on the C-terminal half of GDE

and α-1,6-glucosidase (isomaltase) from the same origin Saccharomyces cerevisiae D-346

showed no significant homology. As the carboxyl group is proposed to be involved in the

catalysis of many glycosyl hydrolases (20), we analyzed the secondary structural features of the

carboxyl residues in the C-terminal half of the yeast GDE by hydrophobic cluster analysis (HCA).


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The oligo-1,6-glucosidase from Bacillus cereus was used as a model enzyme because its catalytic

residues had been identified by X-ray crystallographic analysis (21) (Fig. 2) and it acts on the

α-1,6-glucosidic linkage similar to that of glucosidase of yeast GDE. Indeed, the HCA plot

indicated a significant similarity on the secondary structural features of the C-terminal half of

yeast GDE and oligo-1,6-glucosidase from Bacillus cereus. Among the acidic amino acids,

Asp-1086 and Asp-1147 of yeast GDE showed similar patterns of HCA plot as Asp-206 in

conserved region II and Glu-230 in conserved region III, the two catalytic residues of

oligo-1,6-glucosidase (Fig. 2). Furthermore, these residues were found well conserved among the

amino acid sequences on the C-terminal half of human, rabbit and yeast GDEs. The Asp-1086

and Asp-1147 were hence tagged as the possible candidates for the active sites of

amylo-1,6-glucosidase activity of yeast GDE.

Amino acid substitution by site-directed mutagenesis and the expression and purification of

mutant GDEs - The Asp-535, Glu-564, and Asp-670 on the N-terminal half, and Asp-1086 and

Asp-1147 on the C-terminal half were substituted to their respective amides by site-directed

mutagenesis. Sequence analysis of the mutated GDE genes confirmed the desired mutations

without any second site mutations. All the mutant enzymes were highly expressed and have

the same elution profile on the β-CD Sepharose 6B affinity chromatography as that of the

wild-type enzyme. In addition, all purified mutant GDEs were homogeneous and have the same

molecular mass as the wild-type enzyme.


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Characterization of the mutant GDEs - The glucosidase and transferase activities of mutant

yeast GDEs were determined separately by HPLC using two types of branched cyclodextrin as

substrates, Glc-β-CD and Glc5-β-CD, respectively (Fig. 3 and 4). The purified wild-type enzyme

transfers maltosyl and maltotriosyl residues from one Glc5-β-CD to another to form Glc7-,

Glc8-β-CD and Glc2-, Glc3-β-CD. These branched β-CDs produced by the transferase reaction are

also substrates for the transferase, accordingly Glc-β-CD, Glc2-,-----Glc8-β-CD arise as products

by the transferase action and then Glc-β-CD is hydrolyzed to glucose and β-CD by the

glucosidase action (W in Fig.3-a,b). HPLC profiles of mutants D535N, E564Q, and D670N

gave only a single peak corresponding to substrate Glc5-β-CD (N-M in Fig. 3-a), indicating that

these mutants are deficient in transferase. On the contrary, the HPLC profiles of mutants GDE

D1086N and D1147N showed that in addition to the peak corresponding to the substrate

Glc5-β-CD, products of various branched cyclodextrins, Glc-β-CD, Glc2-β-CD, Glc3-β-CD,

Glc7-β-CD, and Glc8-β-CD were also detected (C-M in Fig. 3-b), inferring an active transferase is

expressed in mutants GDE D1086N and D1147N. In addition, products of the reaction

mixture containing these mutant enzymes indicated no glucose and β-CD (C-M in Fig. 3-b), an

indicative of glucosidase-deficient mutant GDEs.

Using Glc-β-CD as a substrate, the glucosidase activity of GDE was also analyzed by

HPLC. The transferase-deficient mutant enzymes D535N, E564Q and D670N indeed gave

hydrolysis products of glucose and β-CD from the substrate Glc-β-CD as the wild-type enzyme


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does (Fig. 4-a), while the glucosidase-deficient mutant enzymes D1086N and D1147N failed to

hydrolyze the substrate Glc-β-CD (Fig. 4-b).

The HPLC analyses indicate that the mutants retained either the transferase or the

glucosidase activity (Table 1), depending on the site of mutation each possessed. However, the

question remains whether the retained activity of each mutant has varied from that of the

wild-type GDE or not. To know then is to compare the kinetic parameters of each mutant

enzymes and the wild-type enzyme.

Kinetic parameters of mutant GDEs - The glucosidase activity of the transferase-deficient

mutants, D535N, E564Q and D670N, was measured by using Glc-β-CD as a substrate, while the

transferase activity of the glucosidase-deficient mutants, D1086N and D1147N, was measured by

using maltopentaose as a substrate (see EXPERIMENTAL PROCEDURES). As shown in Table 2,

kinetic parameters for the glucosidase activity of transferase deficient mutants were as a same

level that of wild-type enzyme. In addition, Km values for the transferase activity of glucosidase

deficient mutants were also similar to that of wild-type enzyme (D1086N, D1147N, wild-type :

10.0, 10.6, 10.8 mM, respectively). These results indicated that each mutant GDE catalyzes their

respective reaction as the wild-type does without being influenced by the lost of the other

functions.


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DISCUSSION

The five mutants D535N, E564Q, D670N, D1086N and D1147N were produced to

discriminate the individuality of the two catalytic sites of GDE as earlier proposed (8-11). The

identical purification profiles of these mutant enzymes on β-CD Sepharose 6B affinity column

chromatography with the wild type enzyme indicate that the mutant GDEs have the same extent

of binding affinity to β-CD, a substrate analogue, as the wild type. These data suggest that the

mutation incurred did not influence the conformation of the enzymes. As shown in Table 1, the

mutant GDEs possessed either the transferase activity or the glucosidase activity. Mutants

D535N, E564Q and D670N exhibit only the glucosidase activity, while D1086N and D1147N

mutants had only the transferase activity. Moreover, the kinetic parameters of the mutant enzyme

activity were similar to that exhibited by the wild type. To note, Asp-535, Glu-564 and Asp-670

are located at the N-terminal half of the amino acid sequence, while Asp-1086 and Asp-1147 are

at the C-terminal half. We therefore conclude that the active sites of transferase and glucosidase

of the yeast GDE are independent of each other. This study agrees well with the enzyme

inhibitor studies and amino acid sequence analysis of rabbit GDE (8-10) suggesting that the two

activities take place at two distinct catalytic sites of the enzyme molecule. Though Teste et al.

(22) from their analysis of the yeast GDE deletion mutant suggested recently that the C-terminal

half is indispensable for both activities, we believed that it was not the case but instead, the

C-terminal half of GDE might take part only in substrate binding, a function that was lost in the


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deletion mutant due to possible structural changes brought by deletion.

The Asp-535, Glu-564 and Asp-670 residues proposed to comprise the active site of

transferase of yeast GDE are situated in consensus sequences II, III and IV of the N-terminal half,

respectively and so did the two Asp and one Glu (Asp-229, Glu-257, Asp-328) residues identified

as the catalytic residues of cyclodextrin glucanotransferase, CGTase (23-25). The Asp-229 of

CGTase and Asp-549 of rabbit GDE had been confirmed to work as a catalytic nucleophile of

transferase (26, 27). As Asp-535 of yeast GDE corresponds to Asp-229 of CGTase and Asp-549

of rabbit GDE in the amino acid sequence alignment (Fig. 1), possibility exists that Asp-535 may

play the same role on the transferase action. It has been suggested that the GDE transferase is an

α-retaining glycosidase (9) and likewise a CGTase. Therefore, the transferase of yeast GDE

may employ a double displacement mechanism to process α-linked glucose polymers as

demonstrated by CGTase (28).

Spectrochemical analysis of glucosidase reaction revealed that the yeast GDE hydrolyzed

the α-1,6-glucosidic linkage and released a β-anomer of glucose from Glc-β-CD, an inverted

configuration of the expected product (data not shown). Similarly, such α-inverting mechanism

was also observed with the glucosidase of rabbit GDE (9). In the case of α-inverting glycosidase

belonging to the glycosyl hydrolase family 15 (29), the combination of differential labeling and

site-directed mutagenesis or the crystal structure analysis identified two carboxyl residues of

glucoamylase as the catalytic acid/catalytic base (30-32). Hence, Asp-1086 and Asp-1147 of

yeast GDE located at the C-terminal half may play as a general acid catalyst or general base


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catalyst for glucosidase reaction. However, structural conformation of the active sites may differ

between the glucosidase of yeast GDE and glucoamylase, because while the glucoamylase was

inactivated by the modification induced by the inhibitor of water-soluble carbodiimide (33-35),

glucosidase of yeast GDE was not affected at all (data not shown).

In this study, we clearly demonstrated that the transferase and glucosidase of yeast GDE

are well discriminated at two distinct sites of a single polypeptide chain, the transferase being

located at the N-terminal half and the glucosidase at the C-terminal half. (Fig. 5). Since the

catalytic mechanism and primary structural characteristics of transferase of yeast GDE is similar

to the enzymes belonging to the α-amylase family, the N-terminal half of yeast GDE protein may

be folded also in the form of a α/β8-barrel structure. On the other hand, though glucosidase at the

C-terminal half of yeast GDE exhibited a catalytic mechanism (α-inverting) in good agreement

with that of glucoamylase, their primary structural characteristics� , indicating a possible

difference of structural conformation between the two types of glucosidases. This study thereby

provides clear evidence on the distinctiveness of the active sites of the transferase and glucosidase

of yeast GDE and may serve as a basic knowledge for the understanding of the structure-function

relationship of bifunctional glycogen debranching enzymes in depth.


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33. Inokuchi, N., Iwama, M., Takahashi, T., and Irie, M. (1982) J. Biochem. (Tokyo) 91, 125-133.

34. Iwama, M., Ohtsuki, R., Takahashi, T., and Irie, M. (1984) J. Biochem. (Tokyo) 96,

329-336.

35. Koyama, T., Inokuchi, N., Iwama, M., and Irie, M. (1985) J. Biochem. (Tokyo) 97, 633-641.

36. Lemesle-Varloot, L., Henrissat, B., Gaboriaud, C., Bissery, V., Morgat, A., and Mornon, J. P.

(1990) Biochimie 72, 555-574.


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FIGURE LEGENDS

Fig. 1. Consensus sequences of α-amylase family in GDEs The amino acids were numbered

from the N-terminal end. Conserved residues are in bold. Asterisks indicate the target amino acid

residues on the yeast GDE for site-directed mutagenesis. TAA (Taka-amylase, α-amylase from

Aspergillus oryzae), CGTase (cyclodextrin glycosyltransferase from Bacillus circulans), H-GDE

(Human muscle glycogen debranching enzyme), R-GDE (Rabbit muscle glycogen debranching

enzyme), Y-GDE (glycogen debranching enzyme from Saccharomyces cerevisiae D-346).

Fig. 2. Hydrophobic cluster analysis (HCA) plots of oligo-1,6-glucosidase from Bacillus

cereus and C-terminal half of yeast GDE. HCA (36) plots were obtained from the DrawHCA

server (http://www.lmcp.jussieu.fr/~soyer/www-hca/hca-form.html). Numbering starts from the

first amino acid of the mature protein. The regions showing similarity at the HCA level are boxed.

Dark gray circles indicate the catalytic residues of the oligo-1,6-glucosidase from Bacillus cereus

and light gray circles show the position corresponding to the putative active site residues of yeast

GDE. The protein sequences are written on a duplicated α-helical net and the contour of clusters

of hydrophobic residues is automatically drawn. The standard one-letter code for amino acids is

used except for proline, glycine, serine, and threonine, which are represented by ★ , ♦ , �, and �,

respectively.


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Fig. 3. HPLC analysis of the products formed by the action of wild-type and mutant

enzymes on Glc5-β-CD. (a): HPLC profile of Glc5-β-CD treated with mutant D535N (E564Q,

D670N). A mixture (10 µl) containing 10 mM Glc5-β-CD, 25 mM citrate buffer (pH 6.5), and

3 µg of enzyme was incubated at 37 oC for 1 h (5 h for mutant D535N, E564Q, and D670N). Ten

µl of the diluted sample (20 nmol as Glc5-β-CD) was applied to HPLC. (b): HPLC profile of

Glc5-β-CD treated with mutant D1086N (D1147N). Symbols indicate as follows: W, wild-type

enzyme; N-M, mutant enzyme D535 (E564Q, D670N); C-M, mutant enzyme D1086N (D1147N).

Peaks: Glc, glucose; 0, β-CD; numbers 1 - 8 indicate the number of glucose residues on the

branched chain of the cyclodextrin, Glc-β-CD, Glc2-β-CD, Glc3-β-CD, and so on.

Fig. 4. HPLC analysis of the products from Glc-β-CD treated with wild-type or mutant

enzymes. Sample preparation and conditions for enzyme reaction are as described in Fig. 3

except that Glc-β-CD was used as the substrate. (a) HPLC profile of the sugar products released

by the action of mutant enzyme D535N (D564Q, D670N); (b) HPLC profile of the sugar

products released by the action of mutant enzyme D1086 (D1147N). Symbols were as described

in Fig. 3.

Fig. 5. An illustration of the locality of the transferase and glucosidase on the yeast GDE.


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Table 1. Enzymatic properties of the mutant and wild-type GDEs

Activity

Position enzyme Mutation Transferase1) Glucosidase2)

Wild-type none active active

N-terminal half D535N Asp535-Asn inactive active

E564Q Glu 564-Gln inactive active

D670N Asp670-Asn inactive active

C-terminal half D1086N Asp1086-Asn active inactive

D1147N Asp1147-Asn active inactive

1) Activity against Glc5-β-CD as a substrate.

2) Activity against Glc- β-CD as a substrate.


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Table 2. Kinetic parameters of transferase deficient GDE mutants

Glucosidase activity was measured with Glc-cG7 as a substrate

Enzyme Km (mM)

kcat (S-1)

Wild-type 16.3 21.7

D535N 16.6 29.4

E564Q 15.9 21.4

D670N 19.3 26.6


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Akifumi Nakayama, Keizo Yamamoto and Shiro TabataIdentification of the catalytic residues of bifunctional glycogen debranching enzyme

published online May 25, 2001J. Biol. Chem.

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1 identification of the catalytic residues of bifunctional glycogen

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