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INTRODUCTION |
Glucan synthesis catalyzed by glucosyltransferases (GTFs; sucrose
6-glucosyltransferase, EC
2.4.1.5)1 of the oral
streptococci is critical in the development of dental caries. This is
the first step in the formation of dental plaque, which mediates the
aggregation of cariogenic microorganisms, which in turn leads to acid
demineralization of the tooth enamel. Therefore, GTF activity is a
potential up-stream target in the pathological cascade (1, 2).
GTFs catalyze two sequential reactions: the cleavage of sucrose into
fructose and an enzyme-bound glucosyl moiety (sucrase activity), and
the subsequent transfer of the latter to the C-3/C-6 position of the
glucose residue of glucan (transferase activity) or to water (3). In
accordance with these enzymatic properties, GTFs have two relatively
independent structural domains, a catalytic domain comprised of the
N-terminal two-thirds of the protein and a glucan-binding domain
comprised of the remaining one-third (4). Although these domains are
common among GTFs found in streptococci and the sequences of the active
sites are highly conserved (3, 5), each GTF exhibits distinct enzyme
activity that differs in position of glucosyl linkage of product
and/or primer requirement for catalytic activity (1, 3).
Streptococcus mutans (S. mutans), a major
causative organism of human dental caries, produces three GTFs,
i.e. GTF-I (gtfB) and GTF-SI (gtfC),
which catalyze primarily the synthesis of
-1,3-linked water-insoluble glucan and low molecular water-soluble glucan in a
primer-independent manner, and GTF-S (gtfD), which catalyzes the synthesis of
-1,6-linked water-soluble glucan in a
primer-dependent manner (6-8). Disruption analysis of
genes encoding these GTFs revealed that the synthesis of
water-insoluble glucan is essential to the cariogenesis of S. mutans. (9).
GTF-I is composed of 1475 amino acids with a signal peptide of 34 residues at the N-terminal (10). The sequence responsible for sucrose
binding was proposed to be DSIRVDAVD (residues 446-454), and
Asp451 was identified as one of the active centers for
catalytic activity (11, 12). The C-terminal glucan-binding domain
consists of six highly homologous repeating units of approximately
65-amino acid residues each, and deletion analysis has revealed that
the presence of more than three of these units is necessary for GTF to
be able to catalyze glucan synthesis (13). However, details of the
mechanism of catalytic action and structure-function relationships of
GTF remain yet unknown.
In the present study, we paid special attention to the fact that
several enzymes, for example protein kinase C, have been reported to
contain autoinhibitory sequences (14, 15). In addition, peptides
corresponding to the partial sequences of proteins can mimic the
original function of the region and can be used to characterize the
functional domains (16, 17). Based on these facts, we synthesized
19-mer peptides corresponding to various regions of GTF-I and examined
their ability to modulate the catalytic activity of the enzyme. Here,
we demonstrate that a peptide, corresponding to residues 1176-1194,
inhibits GTF-I activity in a sequence-specific manner and three other
peptides, covering residues 902-943, increase GTF-I activity. We
further report the examination of the interaction between GTF-I and the
inhibitory peptide.
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EXPERIMENTAL PROCEDURES |
Peptide Synthesis and Purification--
Peptides were
synthesized by the solid phase Merrifield method (18) in a model 350 peptide synthesizer (Advanced Chemtech, Louisville, KY) and purified by
reverse phase high performance liquid chromatography on a TSKgel column
(ODS-Prep, Tosoh Co., Tokyo, Japan). The molecular masses of peptides
were confirmed by mass spectrometry (JMS.-HX-110A/110A, JEOL Co.,
Tokyo, Japan).
Enzyme Preparation--
GTF-I was prepared from
Streptococcus milleri transformant KSB8 cells expressing the
gtfB gene (19). S. milleri is a noncariogenic oral streptococcus variant that cannot synthesize extracellular glucans
natively and therefore is more suitable than S. mutans for
characterization of GTFs employing recombinant protein expression systems (19, 20). In brief, the cells cultured anaerobically overnight
at 37 °C were collected by centrifugation, washed, and sonicated in
20 mM phosphate buffer (pH 6.0) for isolation of the
cell-associated GTF-I. After centrifugation, the supernatant was used
as crude GTF-I. One unit of enzyme activity was defined as the amount
of enzyme catalyzing the transfer of 1.0 µmol of glucose from sucrose
to glucan/min. For further purification, the crude enzyme solution was
applied to a QAE column (Shimadzu, shim-pack, 1.4 ml) connected to a
Shimadzu high performance liquid chromatography system. The adsorbed
proteins were eluted with a gradient of 0-0.7 M NaCl in 25 mM Tris-HCl (pH 7.5). The fraction eluted at the NaCl
concentration of 0.65 M yielded a GTF-I single band on
SDS-PAGE. For the following assay, the aliquots of the fraction were
dialyzed against 20 mM phosphate buffer (pH 6.0). For the
binding experiment using an optical evanescent resonant biosensor,
which requires a larger amount than for the above purpose, GTF-I was
partially purified using a chromatofocusing column (Amersham Pharmacia
Biotech). After eluting most of the proteins with Polybuffer 74 (Amersham Pharmacia Biotech) and 0.5 M NaCl in 20 mM histidine buffer (pH 6.0), the adsorbed protein
including GTF-I was eluted with 1 M NaCl in 20 mM histidine buffer (pH 6.0) to recover a larger portion of
the enzyme. For the following assay, the aliquots of the fraction were
dialyzed against 50 mM phosphate buffer including 150 mM NaCl (pH 6.0).
Inhibition Assay--
To measure GTF-I activity, we employed a
biochemical assay kit in view of the advantage of being able to examine
sucrose hydrolysis and glucosyl moiety transfer to glucan separately.
For the inhibition assay, 10 milliunits of GTF-I was preincubated in
the presence or absence of 30 µg/ml synthetic peptide in 50 mM phosphate buffer (pH 6.0) at 25 °C for 1 h or
4 °C overnight unless otherwise stated in the figure legends. The
reaction was started by addition of sucrose and, after incubation for
1 h at 37 °C, was stopped by heating at 80 °C for 5 min. The
amounts of glucose and fructose in the reaction mixture were measured
by use of an F-kit (Roche Molecular Biochemicals, Mannheim, Germany).
The amount of fructose in the reaction mixture represents the level of
sucrase activity, and the difference between the amount of free glucose
and that of free fructose in the reaction mixture represents the amount of glucan, the level of total GTF-I activity (21), and the ratio of the
amount of glucan to that of fructose represents the level of glucosyl
transfer activity of GTF-I (transfer activity ratio).
For activity staining, 50 milliunits of GTF-I was preincubated in the
presence or absence of 500 µg/ml synthetic peptide in 50 mM phosphate buffer (pH 6.0) at 4 °C overnight, followed
by addition of SDS-polyacrylamide gel electrophoresis (PAGE)
solubilization buffer to the reaction mixture and SDS-PAGE (22),
without heating of the samples to avoid heat denaturation of the
enzyme. After electrophoresis, the polyacrylamide gel was washed in 20 mM phosphate buffer (pH 6.0) containing 1% Triton X-100
and incubated in sucrose buffer containing 20 mM phosphate
buffer (pH 6.0), 0.125% sucrose, 1% Triton X-100, 2.5 mM
EDTA, and 0.01% Thimerosal, at 25 °C overnight. The synthesized
glucan was observed as a white band on the polyacrylamide gel
(6).
Preparation of Antibody and Western Blot Analysis--
An
anti-GTF-I antibody was raised against a synthetic 25-mer peptide,
CGEFVTDRYGRISYYDANGERVRIN, conjugated to keyhole limpet hemocyanin
(Calbiochem) as described previously (23). This peptide corresponds to
the C-terminal portion of GTF-I (10). 10 milliunits of GTF-I was
subjected to SDS-PAGE, and Western blot analysis using anti-GTF-I
antibody was performed as described previously (23).
Co-precipitation of GTF-I with Peptide 14--
Synthetic peptide
was coupled to support matrix, Affi-Gel 10 gel (Bio-Rad) in 100 mM HEPES (pH 7.0). The gel was preincubated in
phosphate-buffered saline containing 1% bovine serum albumin and 0.5%
Triton X-100 at 4 °C for 1 h, followed by incubation with crude
GTF-I solution at 4 °C overnight in the presence or absence of 500 µg/ml peptide 14. The gel was washed by subjecting it to 10 min of
agitation in phosphate-buffered saline containing 1% Triton X-100,
followed by centrifugation and discard of the supernatant, repeated 5 times. The gel was suspended in SDS-PAGE solubilization buffer and
incubated at 100 °C for 15 min. After centrifugation, the
supernatant was analyzed by Western blot analysis using anti-GTF-I
antibody as described above.
Interaction between GTF-I and Peptide 14 Analyzed by an Optical
Evanescent Resonant Biosensor--
An optical evanescent resonant
mirror cuvette system (IAsys, Affinity Sensors, UK) was used to measure
the interaction of peptide with GTF-I as described (24-26). 5 mg/ml
peptide 14 in acetate buffer (pH 4.5) was immobilized to an aminosilane
cuvette at 25 °C for 10 min following cuvette activation by
bis(sulfosuccinimidyl)suberate (Pierce). The cuvette surface was
inactivated by 1 M ethanolamine/HCl (pH 8.5) and blocked
with bovine serum albumin. The association reaction of GTF-I to the
immobilized peptide was started by addition of the enzyme into the
cuvette, and the dissociation was by replacement of GTF-I with blank
buffer. The traces of the association and dissociation process were
analyzed using Fastfit analysis software supplied with the instrument.
The pseudo first-rate constant, kon, was
obtained for each concentration of the analyte protein. The slope of
the plot of kon against the analyte protein
concentration provides the value for the association rate constant,
ka, and the intercept provides the value for the
dissociation rate constant, kd. The affinity
constant, KD, was calculated from the equation, KD = kd/ka (24-28).
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RESULTS |
Effect of GTF Peptides on GTF-I Activity--
Table
I shows the code numbers of the synthetic
peptides, their amino acid sequences, the corresponding amino acid
positions in GTF-I, their net charge, and their effects on GTF-I
activity. We synthesized 19-mer peptides derived from various GTF-I
regions highly conserved among GTFs of oral streptococci (3, 5) because
we speculated that these regions were of structural and/or functional
importance. The sequences of peptides 32, 33, 1, and 2 are located
upstream of the active center of sucrose hydrolysis, those of peptides
13 and 14 are located in the glucan-binding domain at the C terminus,
and those of the others are located between the active center for
sucrose hydrolysis and the glucan-binding domain (Fig.
1). The role of the region between the
active center and the glucan-binding domain has not yet been reported
on. Some peptides, including the one corresponding to the active site
sequence, were not examined for their effects on GTF-I activity because of their low solubility in buffer solution. Of 22 peptides tested, peptide 14 (residues 1176-1194) had significant effects on GTF-I activity (Table I). Peptide 14 inhibited both sucrase activity and
transferase activity of the enzyme, resulting in a marked reduction in
the amount of glucan. No inhibitory effects of the peptides on
hexokinase and glucose-6-phosphate dehydrogenase activities used for
the biochemical assay were detected even at a higher concentration of
the peptides than that used in Table I. On the other hand, peptides 23, 24, and 31, corresponding to residues 902-943, increased GTF-I
activity by about 20-35%. These peptides primarily affected sucrase
activity, whereas in the presence of these peptides the transferase
activity ratio remained essentially unchanged.

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Fig. 1.
Schematic diagram of the structure of GTF-I
showing the location of the synthetic peptide sequences. The
diagram of the structure of GTF-I and the sequences of the active site
are shown at the top (11). The numbers indicate
the amino acid positions in GTF-I. The aspartic acid
(Asp451) indicated in boldface type is one of
the active centers of sucrose hydrolysis (12). Glucan-binding domain
consists of six highly homologous repeating units: residues 1096-1159,
1160-1223, 1224-1288, 1289-1353, 1354-1418, and 1419-1475 (13).
The domains from which the sequences of the synthetic peptides are
derived are shown at the bottom.
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Lineweaver-Burk Double Reciprocal Analysis of Inhibition by Peptide
14--
Next, we examined the mechanisms of inhibition of GTF-I
activity by peptide 14. GTF-I activity in the presence or absence of
peptide 14 at various concentrations of sucrose was examined, and the
results and deduced Lineweaver-Burk double reciprocal plots are shown
in Fig. 2. Peptide 14 inhibited both
sucrase and GTF-I activities in a noncompetitive manner, indicating
that peptide 14 interacted with enzyme and enzyme-substrate complex
(Fig. 2, D and E). In the absence of peptide 14, about 70-80% of glucose moiety was transferred to glucan, and in the
presence of peptide 14, only 25-30% of glucose moiety was transferred
at any concentration of sucrose (Fig. 2C). The secondary
plots deduced from the double-reciprocal plots yielded an apparent
Ki of 10.5 µM for GTF-I activity (mean
of three independent experiments, S.D. = 4.5). The inhibition of GTF-I
activity by peptide 14 was dose-dependent, and the
concentration of the inhibitor giving 50% inhibition
(IC50) was about 3 µM (Fig. 3).

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Fig. 2.
Kinetic analysis of the inhibitory effect of
peptide 14 on GTF-I activity. Panel A, sucrase activity.
Panel B, GTF-I activity. The activity in the presence
(open circles) and absence (closed circles) of
peptide 14 at the indicated concentration of sucrose was examined.
GTF-I was preincubated with buffer alone or 30 µg/ml peptide 14, and
the reaction was initiated by addition of sucrose of various
concentrations to the reaction mixture. The values are means of
triplicate determinations, and the error bars indicate S.D.
(n = 3). Panel C, transferase activity
ratio. The ratio of glucose moiety to glucan by GTF-I in the presence
(open circles) and absence (closed circles) of
peptide 14 was shown. Panel D, Lineweaver-Burk double
reciprocal plot of sucrase activity. The values in presence (open
triangle) and absence (open rectangle) of peptide 14, which were deduced from the results in panel A, are shown.
Panel E, Lineweaver-Burk double reciprocal plot of GTF-I
activity. The values in presence (open triangle) and absence
(open rectangle) of peptide 14, which were deduced from the
results in panel B, are shown.
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Fig. 3.
Dose-dependent effect of peptide
14 on GTF-I activity. The effects of various concentrations of
peptide 14 on GTF-I activity are shown. GTF-I was preincubated with the
indicated concentrations of peptide, and the activity was measured as
described above with 10 mM sucrose. The values are means of
triplicate determinations, and the error bars indicate S.D.
(n = 3).
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Effect of Peptide 14 on Purified GTF-I--
To rule out the
possible involvement of other putative components which might interact
with peptide 14 to affect GTF-I activity, we examined the effect of
peptide 14 on purified GTF-I. The crude enzyme solution was applied to
an anion exchange HPLC, and the fraction eluted at the NaCl
concentration of 0.65 M yielded a GTF-I single band on
SDS-PAGE (Fig. 4A), although
GTF-I activities were eluted broadly at NaCl concentrations ranging
from 0.25 M to 0.7 M (data not shown). We used
this fraction as purified GTF-I to examine the effect of peptide 14, and peptide 14 inhibited the enzyme activity to a similar degree as
shown with crude enzyme (Figs. 3 and 4B).

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Fig. 4.
Effects of peptide 14 on purified GTF-I.
Panel A, Coomassie Brilliant Blue staining of a fraction
separated by QAE column. The fraction eluted at the NaCl concentration
of 0.65 M was subjected to SDS-PAGE, and the acrylamide gel
was stained with Coomassie Brilliant Blue. The arrow
indicates the relative molecular weight of GTF-I, 150,000, as confirmed
by Western blot analysis using anti-GTF-I antibody. The bars
to the right indicate the positions of marker proteins
(Bio-Rad) and their molecular weights (myosin, 201,000;
-galactosidase, 120,000; bovine serum albumin, 85,000, ovalbumin,
47,000). These molecular weight markers are prestained, and their
apparent molecular weights were determined by calibration against
native proteins. Panel B, the effects of the indicated
concentrations of peptide 14 on purified GTF-I are shown. The
concentration of sucrose used was 10 mM. The values are
means of triplicate determinations, and the error bars
indicate S.D. (n = 3).
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Effect of Scramble Peptides of Peptide 14--
Furthermore, we
synthesized two scramble peptides, pep14/sc1 and pep14/sc2, to evaluate
the sequence specificity of the inhibition (Fig.
5A). The scramble peptides
have the identical amino acid compositions as that of peptide 14, but
the sequences are randomized (29). As shown in Fig. 5B,
neither pep14/sc1 nor pep14/sc2 inhibited GTF-I activity. This result
excluded the possibility that the inhibition was because of the net
charge of the peptide and indicated that peptide 14 inhibited the GTF-I
activity in a sequence-specific manner.

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Fig. 5.
Effects of scramble peptides corresponding to
peptide 14 on GTF-I activity. Panel A, the sequences of the
scramble peptides (see text). Panel B, the effects of
scramble peptides corresponding to peptide 14 on GTF-I activity. GTF-I
was preincubated with buffer or 50 µg/ml peptide 14 or a scramble
peptide. The activity was measured as above with 10 mM
sucrose. The values are means of triplicate determinations, and the
error bars indicate S.D. (n = 3).
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Direct Molecular Interaction of GTF-I with Peptide 14--
We
further examined the effects of peptide 14 by activity staining. The
enzyme preincubated in the presence or absence of synthetic peptide was
subjected to SDS-PAGE without heating of the samples, and after
electrophoresis, the polyacrylamide gel was incubated in sucrose
buffer. As shown in Fig. 6A,
peptide 14 inhibited glucan synthesis by GTF-I on the polyacrylamide
gel. pep14/sc1 had no effect on enzyme activity, consistent with the results in Fig. 5. In addition, we examined the interaction of GTF-I
and peptide 14 using the support gel (Fig. 6B). GTF-I was co-precipitated with peptide 14 coupled to the gel as revealed by
Western blot analysis, whereas it was not co-precipitated with pep14/sc2. Furthermore, addition of excess free peptide 14 in the
reaction solution resulted in the disappearance of co-precipitated GTF-I. Moreover, the interaction between GTF-I and peptide 14 was
confirmed using an optical evanescent resonant mirror cuvette system
(IAsys). GTF-I of various concentrations from 100 nM to 1 µM was examined for association/dissociation to peptide
14 immobilized on the aminosilane cuvette. We did not add the substrate
into the enzyme solution in this experiment because the glucan product caused aggregation of the enzyme and would interfere with the analysis.
As shown in Fig.
7, the
interaction was dependent on the enzyme concentration. The traces of
the association and dissociation were analyzed using Fastfit analysis
software. Analysis revealed that the interaction was biphasic and
yielded first and second rate constants, kon(1)
and kon(2). ka,
kd, and KD obtained from the results of kon(1) were 8.2 × 104 M
1s
1, 9.8 × 10
3 s
1, and 120 nM,
respectively, and 8.3 × 103
M
1s
1, 2.3 × 10
3 s
1, and 277 nM,
respectively, from kon(2). These results
strongly indicate that GTF-I and peptide 14 directly interacted in a
biphasic manner, although the critical region of interaction of both
was not identified, and that this complex could not be separated by sodium dodecyl sulfate during electrophoresis.

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Fig. 6.
The direct interaction of GTF-I with peptide
14. Panel A, the effects of peptide 14 as determined by
activity staining. GTF-I was preincubated with buffer (lane
1), or peptide 14 (lane 2), or pep14/sc1 (lane
3), and subjected to SDS-PAGE as described under "Experimental
Procedures." Bands of synthesized glucan could be visualized after
incubation of polyacrylamide gel in sucrose buffer. The
arrow indicates the relative molecular weight of GTF-I,
150,000, as confirmed by Western blot analysis using anti-GTF-I
antibody. The bars to the right indicate the
positions of marker proteins (Bio-Rad). The molecular weight markers
are identical to those in Fig. 4. The results are representative of
four independent experiments. Panel B, co-precipitation of
GTF-I with peptide 14. GTF-I was incubated with peptide 14 (lanes
1 and 3) or pep14/sc2 (lane 2) coupled to
the support gel in the absence (lanes 1 and 2) or
presence (lane 3) of free peptide 14. The supernatant eluted
from the washed gel was subjected to Western blot analysis. The results
are representative of three independent experiments.
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Fig. 7.
The interaction between GTF-I and immobilized
peptide 14 on aminosilane surface cuvette. Peptide 14 was
immobilized on aminosilane cuvette of an optical evanescent resonant
mirror cuvette system. GTF-I of the indicated concentration was added
into the cuvette and was replaced with blank buffer at the time
indicated by the arrowhead. Association/dissociation of
GTF-I to immobilized peptide 14 was examined as described under
"Experimental Procedures."
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DISCUSSION |
In this report, we have demonstrated that peptides derived from
GTF-I affected the enzyme activity; those corresponding to residues
902-943 (peptides 23, 24, and 31) elevated GTF-I activity, and the one
corresponding to residue 1176-1194 (peptide 14) inhibited GTF-I
activity. Peptide 14 directly interacted with GTF-I in a biphasic
manner to inhibit the enzyme activity (Figs. 4, 6, and 7), and this
inhibition was noncompetitive with the substrate sucrose (Fig. 2).
Furthermore, inhibition was in a sequence-specific manner, and the net
charge of the peptide does not explain the inhibition because neither
peptides with similar net charge (Table I) nor scramble peptides (Fig.
5) were effective.
Several proteinaceous inhibitors of
-amylase, a GTF-related enzyme,
have been identified, for example tandemstat (30). It is interesting
that the complex of tandemstat and amylase cannot be separated by
sodium dodecyl sulfate (30), similar to the case for peptide 14 and
GTF-I (Fig. 6A). However, no marked amino acid sequence
homology was found between tandemstat and peptide 14, and peptide 14 does not contain the consensus sequence WRY, which is typical of the
amylase inhibitors that bind to the catalytic site (31).
The residues 1176-1194, represented by peptide 14, are located in the
second repeating unit in the glucan binding domain. Because the mode of
inhibition by peptide 14 is noncompetitive (Fig. 2) and because the
peptide is tightly bound to the enzyme once complexed (Fig. 6), this
segment may play a crucial role in regulation of the enzyme activity.
The binding experiments using IAsys demonstrated that the
enzyme-peptide binding processes were composed of complex
molecular-to-molecular interactions (Fig. 7). The double rate constants
seem to represent intrinsic biomolecular interactions, such as some
conformational changes occurring after the first binding or double
binding site on the enzyme, rather than the steric hindrance (27, 28). We suppose that the second rate binding may be important for inhibition compared with the Ki and IC50 values
(Figs. 2, 3, and 7) although we do not have a rationale for the
difference of these values at present. However, we have cogitated that
the most possible reason for the difference is the lack of the
substrate sucrose in the binding experiment using IAsys. The presence
of sucrose should have some influences on the enzyme-peptide
interactions in the noncompetitive inhibitory reactions (Fig. 2). Also,
the difference could be because of immobilization of the peptide.
How the ability of the peptide to inhibit GTF-I activity is related to
the function of the region corresponding to it in the molecule remains
to be elucidated. However, residues 902-943 are located in the most
homologous region among streptococcal GTFs (3) and also have sequence
similarity to barley
-amylase (approximately 70% similarity in this
region) (10, 32, 33), suggesting its functional importance. Therefore,
it is possible that interactions of the synthetic peptides with the
enzyme region in a homophilic manner may have resulted in modulated
activity. Alternatively, these peptides may have interfered with
conformational changes and/or intramolecular interaction induced in GTF
catalytic process by mimicking the function of the enzyme region.
Our results demonstrate the possibility that the use of a peptide
derived from the enzyme would be effectual to modulate enzyme activity,
as well as to identify the functional domains necessary for
protein-protein interactions as previous studies have shown (16, 17).
We expect that these peptides, besides clinical applications, could be
strong tools to elucidate the structure-function relationships of
GTF-I, which would provide us with clues to clarify why GTF is
responsible for
-1,3/1,6-glucosidic linkage, whereas most
-glucosidase, including amylase, are responsible for
-1,4/1,6-glucosidic linkage. For both these purposes, we would also
like to emphasize that the effectiveness of the peptide may be further
improved by additional modifications such as acetylation, amidation,
and amino acid substitution/addition.