1 identification of the catalytic residues of bifunctional glycogen
TRANSCRIPT
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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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REFERENCES
1. Lee, E. Y. C. and Whelan, W. J. (1971) “Glycogen and starch debranching enzyme” in The
Enzymes (Boyer, P. D., ed.), 3rd ed., vol. 5, pp. 191-234, Academic Press, New York.
2. Taylor, C., Cox, A. J., Kernohan, J. C., and Cohen, P. (1975) Eur. J. Biochem. 51, 105-115.
3. Bates, E. J., Heaton, G. M., Taylor, C., Kernohan, J. C., and Cohen, P. (1975) FEBS Lett. 58,
181-185.
4. Van Hoof, F. and Hers, H. G. (1967) Eur. J. Biochem. 2, 265-270.
5. Chen, Y. T. and Burechell, A. (1995) In The Metabolic and Molecular Bases of Inherited
Disease. (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.) pp 935-965,
McGraw-Hill, NewYork.
6. Lee, E. Y., Carter, J. H., Nielsen, L. D., and Fischer, E. H. (1970) Biochemistry 9, 2347-2355.
7. Tabata, S. and Hizukuri, S. (1992) Eur. J. Biochem. 206, 345-348.
8. Gillard, B. K. and T. E. Nelson. (1977) Biochemistry 16, 3978-3987.
9. Liu, W., Madsen, N. B., Braun, C., and Withers, S. G. (1991) Biochemistry 30, 1419-1424.
10. Liu, W., De Castro, M. L., Takrama, J., Bilous, P. T., Vinayagamoorthy, T., Madsen, N. B.,
and Bleackley, R. C. (1993) Arch. Biochem. Biophys. 306, 232-239.
11. Nakayama, A., Yamamoto, K., and Tabata, S. (2000) Protein Expr. Purif. 19, 298-303.
12. Yang, B. Z., Ding, J. H., Enghild, J. J., Bao, Y., and Chen, Y.-T. (1992) J. Biol. Chem. 267,
9294-9299.
by guest on January 30, 2018http://w
ww
.jbc.org/D
ownloaded from
� ��
13. Sanger, F., Niclen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74,
5463-5467.
14. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M.
D., Fujimoto E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150,
76-85.
15. Tabata, S. and Dohi, Y. (1992) Carbohydr. Res. 230, 179-183.
16. Kunst, A., Draeger, B., and Ziegenhorn, J. (1984) Methods of enzymatic analysis, 3rd ed.
(Bergmeyer, H. U., ed) vol. VI, pp. 163-172, Verlag Chemie, Weinheim.
17. Henrissat, B. and Davies, G. (1997) Curr. Opin. Struct. Biol. 7, 637-644.
18. Svensson, B. (1994) Plant Mol. Biol. 25, 141-157.
19. Jespersen, H. M., MacGregor, E. A., Henrissat, B., Sierks, M. R. and Svensson, B (1993) J.
Protein Chem. 12, 791-805.
20. Ly, H. D. and Withers, S. G. (1999) Annu. Rev. Biochem. 68, 487-522.
21. Watanabe, K., Hata, Y., Kizaki, H., Katsube, Y., and Suzuki, Y. (1997) J. Mol. Biol. 269,
142-153.
22. Teste, M. A., Enjalbert, B., Parrou, J. L., and Francois, J. M. (2000) FEMS Microbiol. Lett.
193, 105-110.
23. Klein, C. and Schulz, G. E. (1991) J. Mol. Biol. 217, 737-750.
24. Klein, C., Hollender, J., Bender, H., and Shulz, G. E. (1992) Biochemistry 31, 8740-8746.
25. Nakamura, A., Haga, K., Ogawa, S., Kuwano, K., Kimura, K., and Yamane, K. (1992) FEBS
by guest on January 30, 2018http://w
ww
.jbc.org/D
ownloaded from
� ��
Lett. 296, 37-40.
26. Mosi, R., He, S., Uitdehaag, J., Dijkstra, B. W., and Withers, S. G. (1997) Biochemistry 36,
9927-9934.
27. Braun, C., Lindhorst, T., Madosen, N. B., and Withers, S. G. (1996) Biochemistry 35,
5458-5463.
28. Uitdehaag, J. C. M., Mosi, R., Kalk, K. H., Veen, B.A., Dijkhuizen, L., Withers, S. G., and
Dijkstra, B. W. (1999) Nat. Struct. Biol. 6, 432-436.
29. Henrissat, B. (1991) Biochem. J. 280, 309-316.
30. Sierks, M. R., Ford, C., Reilly, P. J., and Svensson, B. (1990) Protein Eng. 3, 193-198.
31. Svensson, B., Clarke, A. J., Svendsen, I., and Moller, H. M. (1990) Eur. J. Biochem. 188,
29-38.
32. Harris, E. M. S., Aleshin, A. E., Firsov, L. M., and Honzatko, R. B. (1993) Biochemistry 32,
1618-1626.
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.
10.1074/jbc.M102192200Access the most updated version of this article at doi:
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