Kinetic analysis and mechanism on the inhibition of chlorogenic acid and its components against porcine pancreas α-amylase isozymes I and II

Article abstract of DOI:10.1021/jf9017383

Chlorogenic acid (5-caffeoylquinic acid, 5-CQA) is a kind of polyphenol and is richly included in green coffee beans. The inhibitory effects of 5-CQA and its components, caffeic acid (CA) and quinic acid (QA), on the two porcine pancreas α-amylase (PPA) i

Full text of DOI:10.1021/jf9017383

9218 J. Agric. Food Chem. 2009, 57, 9218–9225
DOI:10.1021/jf9017383
Kinetic Analysis and Mechanism on the Inhibition of
Chlorogenic Acid and Its Components against Porcine
Pancreas r-Amylase Isozymes I and II
†
,‡
,†
*
Y
USAKU
NARITA
AND
K
UNIYO
I
NOUYE
†
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
‡
Sakyo-ku, Kyoto 606-8502, Japan, and R&D Center, UCC Ueshima Coffee Co, Ltd., 3-1-4 Zushi,
Takatsuki-shi, Osaka 569-0036, Japan
Chlorogenic acid (5-caffeoylquinic acid, 5-CQA) is a kind of polyphenol and is richly included in
green coffee beans. The inhibitory effects of 5-CQA and its components, caffeic acid (CA) and
quinic acid (QA), on the two porcine pancreas R-amylase (PPA) isozymes, PPA-I and PPA-II, were
investigated using p-nitrophenyl-R- -maltoside as substrate at pH 6.9 and 30 °C. The inhibition
D
potencies of the respective inhibitors against both PPA isozymes were almost the same and in the
order of 5-CQA > CA . QA. Their IC50 values were 0.07-0.08 mM, 0.37-0.40 mM, and 25.3-26.5
mM, respectively. The inhibition mechanisms of 5-CQA and CA were investigated by kinetic
0
analyses, and the inhibitor constants K
i
and K
i
(for the free enzyme and enzyme-substrate
complex, respectively) were determined. It was indicated that 5-CQA and CA showed mixed-type
0
inhibition with K > K against both PPA-I and PPA-II. The binding of PPA-I or PPA-II with 5-CQA or
i
i
CA was all exothermic and enthalpy-driven. QA is a poor inhibitor, and its inhibitory mode was
unique and hardly analyzed by a simple Michaelis-Menten-type interaction between the enzyme
and inhibitor. However, it was shown that the inhibitory activity of CA was enhanced 5 times by
ester-bond formation with QA in the form of 5-CQA. These results provide us with significant hints
for the development of R-amylase inhibitors useful for the prevention of diabetes and obesity.
KEYWORDS: R-Amylase inhibitor; caffeic acid; chlorogenic acid; enzyme kinetics; mixed-type
inhibition
INTRODUCTION
tion, virus-I integrase inhibition, DNA methylation inhibition,
and so forth (9-11).
Chlorogenic acids (CGAs) refer to a family of esters between
quinic acid and one or plural cinnamic acid derivatives such as
caffeic, ferulic, and p-coumaric acids. They are richly contained
in green coffee beans, and 34 kinds of CGAs were reported in
green coffee beans (1, 2). The contents of CGAs in green coffee
beans are 3.5-7.5% (w/w-dry matter) for Coffea arabica and
Porcine pancreas R-amylase (EC 3.2.1.1, hereafter abbreviated
as PPA) is an endoglucanase that catalyzes the hydrolysis of
R-1,4-glycosidic linkages in starch, amylose, amylopectin, and
glycogen (12). PPA is composed of 496 amino acid residues and
shows 83% homology with human pancreas R-amylase (13-15).
Two active components were separated from a crystalline pre-
paration of PPA in anion-exchange chromatography using
DEAE-gels, and the component rapidly eluted was designated
as PPA-1, and that slowly eluted was PPA-II (16, 20). These
isozymes are secreted in about equal amounts into pancreatic
juice (16). The amino acid sequence of PPA-I is known (15, 17),
whereas only a partial amino acid sequence has been reported for
PPA-II (18). PPA-I and PPA-II have almost the same molecular
mass (19). The optimum pH values and temperatures for both
isozymes in starch hydrolysis activity were similar and are 6.9 and
7.0-14.0% (w/w-dry matter) for Coffea canephora (3, 4). CGAs
include three main classes which are caffeoylquinic acids,
dicaffeoylquinic acids, and feruloylquinic acids (3, 4). Their
nomenclature is based on the IUPAC numbering system, and
5-caffeoylquinic acid (5-CQA) is generally called chlorogenic
acid (5). The level of 5-CQA is more than 50% (w/w-dry matter)
of total CGAs in green coffee beans (3, 4). Figure 1 shows the
structures of 5-CQA and the components, caffeic acid (CA) and
quinic acid (QA). CGAs are widely distributed in other plants
such as yacon, burdock, prune, pear, potato, and sweet
potato (6-8). Many investigators have reported that CGAs have
various biological activities, such as antioxidant activity, anti-
mutagenicity, suppression of cancer cells, melanin synthesis
inhibition, matrix metalloproteinase inhibition, tyrosinase inhibi-
5
3 °C, respectively (20). Their isoelectric points are slightly
different and are 6.5 and 6.1, respectively (20).
R-Amylase inhibitors seem to be effective for the prevention
and therapy of metabolic syndromes such as type II diabetes and
obesity in controlling the elevation of plasma blood glucose levels
by delaying postprandial carbohydrate digestion and absorption.
R-Amylase inhibitor from white beans (Phaseolus vulgaris) was
*
To whom correspondence should be addressed. Tel: þ81-75-753-
6
266. Fax: þ81-75-753-6265. E-mail: inouye@kais.kyoto-u.ac.jp.
pubs.acs.org/JAFC
Published on Web 09/16/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 19, 2009 9219
were stained with Simplyblue safe stain buffer for 1 h, and their molecular
weights were estimated using Novex Sharp Unstained Protein Standard
(
Invitrogen). BN-PAGE was also carried out with the system and
reagents of Invitrogen. Protein samples were mixed with NativePAGE
sample buffer, NativePAGE 5% G-250 sample additive, and 10%
n-dodecyl-β-
PAGE 4-16% Bis-Tris gel in the Xcell Surelock Mini-Cell at 25 °C for
20 min at 150 V. The anode and cathode buffers were NativePAGE
D-maltoside. The samples were electrophoresed on a Native-
1
running buffer and that containing NativePAGE cathode buffer additive,
respectively.
Hydrolysis of G -pNP by PPA. It is known that PPA hydrolyzes
2
2
G -pNP at one position to produce p-nitrophenol (pNP) and maltose (26).
Hydrolysis of G -pNP by 0.8 μM PPA (PPA-I or PPA-II) in 20 mM
2
sodium phosphate buffer (pH 6.9, buffer B) containing 25 mM NaCl was
measured continuously following the increase in absorbance at 400 nm due
to pNP. The amount of pNP was evaluated using the molar absorption
-1
-1
coefficient of 9.47 mM cm at pH 6.9 (27). The molecular activity (kcat
and Michaelis constant (K ) were calculated from Hanes-Woolf plots
and Lineweaver-Burk plots (28, 29).
)
m
Inhibition of PPA by 5-CQA, CA, and QA. Inhibitory activities of
-CQA, CA, and QA against PPA were examined spectrophotometrically
Figure 1. Structures of chlorogenic acid, caffeic acid, and quinic acid.
5
2
under the conditions used for the PPA activity measurement with G -pNP
as substrate. Ten microliters of 40 μM PPA and 430 μL of various
concentrations of the inhibitor ([5-CQA] = 0.0232-4.65 mM, [CA] =
reported to reduce glycemia in both nondiabetic and diabe-
tic animals and reduced the intake of food and water (21).
R-Amylase inhibitor isolated from chestnut astringent skin ex-
tract was reported to suppress the risein plasma glucose level after
boiled-rice loading in a dose-dependent manner in humans (22).
Various R-amylase inhibitors such as acarbose (19, 23) and
protein R-amylase inhibitor from wheat kernel (24) are discussed
in the inhibition mechanism against PPA.
0
.116-4.65 mM, and [QA] = 5.81-11.6 mM) were mixed first and
incubated for 30 min at a temperature of 20-40 °C. The hydrolysis was
initiated by adding 60 μL of various concentrations (33.3-100.0 mM) of
G -pNP to the enzyme-inhibitor mixture. The initial concentrations of
2
2
PPA, 5-CQA, CA, QA, and G -pNP in the reaction mixture were 0.80 μM,
0.020-4.00 mM, 0.10-4.00 mM, 5.0-60.0 mM, and 4.0-12.0 mM,
respectively. All measurements were performed in triplicate. Relative
activity (%) in the presence of inhibitor was calculated by eq 1 using the
initial velocities v and v in the absence and presence of the inhibitor,
e i
respectively.
5
-CQA, CA, and QA have already been reported to have
inhibitory activity against commercially available crude PPA
preparations in starch hydrolysis (25). However, the inhibitory
activity against the purified PPA-I and PPA-II preparations has
not yet been investigated. Furthermore, the inhibition mechanism
has not been reported. In the present article, we describe the
kinetic and thermodynamic analyses of the inhibitory effects of
relative activity ð%Þ ¼ 100ꢀðν
i
=ν
e
Þ
ð1Þ
The IC50 value was defined as the inhibitor concentration required for
giving the relative activity of 50%. The type of inhibition was determined
5
-CQA, CA, and QA on PPA-I and PPA-II activities and discuss
by Hanes-Woolf plots and Lineweaver-Burk plots. The inhibitor con-
0
their inhibitory mechanisms. We also provide some insights into
the nutritional significance of the inhibitors and their preventive
effects against diabetes and obesity.
stants (K
i
and K
i
) were defined using the following general equation (eq 2)
for mixed-type inhibition (28, 29):
ν
i
¼ Vmax½Sꢁ=fK
m
ð1 þ ½Iꢁ=K
i
Þg þ f½Sꢁð1 þ ½Iꢁ=K
i
0Þg
ð2Þ
In eq 2, [S] and [I] are the initial concentrations of substrate and inhibitor,
MATERIALS AND METHODS
respectively; Vmax is the maximum velocity observed under the conditions,
Materials. p-Nitrophenyl-R-
purchased from Calbiochem (San Diego, CA). Pancreatin (lot M1P6865),
chlorogenic acid hemihydrate (lot M7M4404), CA (lot M4F5063), -(-)-
2
D-maltoside (lot B76297-1, G -pNP) was
m i
[S] . K , in the absence of inhibitor; K is the inhibitor constant or the
dissociation constant for the enzyme-inhibitor (EI) complex into the
D
0
inhibitor (I) plus enzyme (E); and
K
i
is that for the enzy-
QA (or QA, lot M5M5664), and all other chemicals were of reagent grade
me-substrate-inhibitor (ESI) complex into I plus the enzyme-substrate
and were purchased from Nacalai Tesque (Kyoto, Japan).
(
ES) complex.
Purification of PPA-I and PPA-II. PPA-I and PPA-II were purified
according to the procedures previously reported (24) with some modifica-
tions. One gram of pancreatin was dissolved in 50 mL of 20 mM Tris-HCl
buffer (pH 8.3, buffer A), to which 33 mL of chilled acetone was added,
followed by centrifugation at 10,000g for 30 min at 4 °C. Acetone was
further added to the supernatant to a final concentration of 67%, and the
precipitates were collected by centrifugation. The precipitates were dis-
solved in buffer A and applied to anion-exchange HPLC on a TSKgel
BioAssist Q column [10.0 mm (inner diameter) ꢀ 10.0 cm] (Tosoh, Tokyo,
Japan) equilibrated with buffer A at 27 °C. A linear gradient was generated
from 0 M to 50 mM NaCl at time 20.0 min over 40.0 min at a flow-rate of
o
The standard enthalpy change (ΔH ) for the binding of the enzyme
(
PPA-I and PPA-II) with the inhibitor (5-CQA and CA) was determined
o
0
from the slope (ΔH /R) of the plots of 1/K
Hoff plots). The standard Gibbs energy change (ΔG ) and standard
entropy change (ΔS ) for the binding of PPA with the inhibitor were
i
and 1/K
i
against 1/T (van’t
o
o
determined according to the following equations:
o
o
0
i
ΔG ¼ -RT lnð1=K Þ or ΔG ¼ -RT lnð1=K Þ
i
ð3Þ
ð4Þ
o
o
o
ΔS ¼ ðΔH - ΔG Þ=T
1.0 mL/min. Absorption was measured with a Hitachi U-2910 spectro-
where R is the gas constant, and T is the temperature in Kelvin units.
meter (Tokyo).
0
i i
Hereafter, the thermodynamic parameters calculated based on K and K
Sodium Dodecyl Sulfide-Polyacrylamide Gel Electrophoresis
SDS-PAGE) and Blue Native (BN)-PAGE. SDS-PAGE was
o
0
were designated with the suffices of K and K , respectively, as ΔG Ki,
i i
(
o
ΔG Ki
0
, and so forth.
carried out as follows according to the manual with the system and
reagents supplied by Invitrogen (Carlsbad, CA). Protein samples were
reduced by treatment with NuPAGE LDS sample buffer and NuPAGE
reducing agent at 70 °C for 10 min. The samples were electrophoresed on a
NuPAGE Novex 4-12% Bis-Tris gel with NuPAGE MES SDS running
buffer in the Xcell SurelockMini-Cell at 25 °C for 35 min at 200 V. Proteins
RESULTS
Purification of PPA Isozymes. Two PPA fractions were eluted
at 32.6 and 56.5 min in anion-exchange HPLC on a TSKgel
BioAssist Q column. According to the nomenclature given

9220 J. Agric. Food Chem., Vol. 57, No. 19, 2009
Narita and Inouye
Figure 3. Inhibitoryeffects of 5-CQA, CA, and QAagainstPPA-IandPPA-
II. PPA inhibitory activity assays were preformed in buffer B containing
2
0
5
5 mM NaCl at pH 6.9 and 30 °C. The initial concentration of PPA was
.80 μM and that of G -pNP was 8.0 mM. The initial concentrations of
-CA, CA, and QAwereintherange of 0-4mM, 0-4 mM, and0-60 mM,
2
respectively. (Panel A) Inhibition curves of 5-CQA against PPA-I activity
Figure 2. Purification of PPA-I and PPA-II by anion-exchange HPLC with
TSKgel BioAssist Q and electrophoretic analyses of the purified PPA
isozymes. (Panel A) A typical HPLC chromatogram of crude PPA
(
O), 5-CQA against PPA-II (b), CA against PPA-I (4), and CA against
PPA-II (2). (Panel B) Inhibition curves of QA against PPA-I activity (0)
and QA against PPA-II (9). Each point represents the mean of triplicate
experiments.
(
precipitates formed from pancreatin by 40-67% acetone). Fractions
elutedaround32.6 and56.5 min werecollected aspurified PPA-I and PPA-
II preparations. (Panel B) SDS-PAGE of purified PPA-I and PPA-II. Lane
M, molecular-weight markers (Novex Sharp Protein Standard, Invitrogen).
Lane 1, crude PPA containing 7.5 μg protein; lane 2, purified PPA-I
containing 5.0 μg protein; and lane 3, purified PPA-II containing 5.0 μg
protein. (Panel C) BN-PAGE of purified PPA-I and PPA-II. Lane 1,
purified PPA-I containing 5.0 μg protein and lane 2, purified PPA-II
containing 5.0 μg protein. In SDS-PAGE and BN-PAGE, the cathode
and anode were placed on the bottom and top sides of the PAGE plates,
respectively.
PPA-II-catalyzed hydrolysis of G -pNP at pH 6.9 and 30 °C.
2
At the enzyme concentration of 0.80 μM and the substrate
concentration of 8.0 mM, the v values of PPA-1 and PPA-II
e
were determined to be 20.5 (1.3 and 23.1 ( 1.9 nM/s, res-
pectively. The relative activities of PPA-I and PPA-II de-
creased with increasing 5-CQA concentration in a similar man-
ner, and the IC50 values against PPA-I and PPA-II were 0.08 (
0.02 and 0.07 ( 0.02 mM, respectively (Figure 3A). The complete
inhibition of PPA-I was observed at 1.5 mM 5-CQA, and that of
PPA-II was at 2.0 mM 5-CQA. The relative activities of PPA-I
and PPA-II decreased also in a similar manner with increasing the
CA concentration, and the IC50 values against PPA-I and PPA-II
were 0.40 ( 0.03 and 0.37 ( 0.04 mM, respectively (Figure 3A).
Almost all activities of PPA-I and PPA-II were lost in the
presence of 4 mM CA. The curves showing the dependence of
the relative activities of PPA-I and PPA-II on the QA concentra-
tion were substantially the same, although considerably different
from those on the 5-CQA and CA concentrations (Figure 3B).
The activities higher than 80% remained for both isozymes even
in the presence of 15 mM QA, while they decreased sharply when
the QA concentration increased from 15 to 35 mM. The IC50
values of QA against PPA-I and PPA-II were 26.5 ( 1.8 and
previously (16, 20), the respective fractions were assigned as
PPA-I and PPA-II fractions (Figure 2A). The collected fractions
were used for further analyses. Each of the PPA-I and PPA-II
preparations showed a single band corresponding to 55.4 kDa on
SDS-PAGE (Figure 2B) and also a single band on BN-PAGE
(
Figure 2C). The concentrations of PPA-I and PPA-II were
determined using the molar absorption coefficient at 280 nm
of 0.138 μM- cm calculated using the molecular mass of
1
-1
55.4 kDa and absorbance value at 280 nm (A280, 1 cm = 2.5) and at
the protein concentration of 1.0 mg/mL (19).
Inhibition of PPA by 5-CQA, CA, and QA. Figure 3 shows
the inhibition by 5-CQA, CA, and QA of the PPA-I- and

Article
J. Agric. Food Chem., Vol. 57, No. 19, 2009 9221
Figure 4. Temperature dependence of the inhibitory activity of 5-CQA,
CA, and QA against PPA-I and PPA-II. PPA-I activity in the presence of
5
-CQA (O), CA (4), and QA (0) were performed in buffer B containing
5 mM NaCl at pH 6.9 in the temperature range of 20-40 °C. The initial
2
concentrations of PPA-I, G
2
-pNP, 5-CQA, CA, and QA were 0.80 μM,
8.0 mM, 0.1 mM, 0.3 mM, and 25 mM, respectively. PPA-II activity in the
presence of 5-CQA (b), CA (2), and QA (9) were performed under the
same conditions as those for PPA-I. Each point represents the mean of
triplicate experiments.
2
5.3 ( 0.7 mM, respectively, and the complete inhibition of PPA-
I was given with 35.0 mM QA, and that of PPA-II was with
0.0 mM QA. Comparing the IC50 values of 5-CQA, CA, and
4
QA, the orders of the inhibitory activities of the inhibitors were
the same for PPA-I and PPA-II and were 5-CQA > CA . QA.
When Δv (= v - v ) was plotted against the concentration of
e
i
Figure 5. Hanes-Woolf plots for the PPA-I -catalyzed hydrolysis of
inhibitors, 5-CQA and CA, the curves of the plots were similar to
2
G -pNP. PPA-I activity was measured in the presence of 5-CQA (panel
a standard profile of Michaelis-Menten kinetics and the plots of
A) and CA (panel B) under the same conditions as those shown in Figure 2.
[
(
I]/Δv against [I] (Hanes-Woolf plots) and 1/Δv against 1/[I]
(
(
Panel A) The 5-CQA concentration in the reaction mixture: 0 M (O), 50 μM
2), 100 μM (9), and 200 μM ((). (Panel B) The CA concentration in the
Lineweaver-Burk plots) gave straight lines (data not shown),
indicating that each inhibitor binds stoichiometrically with each
PPA isozyme at a molar ratio of 1:1. However, the dependence of
Δv on QA concentration did not show Michaelis-Menten-type
profiles for PPA-I and PPA-II (data not shown), suggesting that
the inhibitory mechanism of QA against PPA might be unique
and different from that of 5-CQA and CA.
reaction mixture: 0 M (O), 100 μM (2), 200 μM (9), and 300 μM ((). Each
point represents the mean of triplicate measurements.
of G -pNP in the absence and presence of various concentrations
2
of 5-CQA and CA at pH 6.9 and 30 °C. Figure 5 shows the
Hanes-Woolf plots for the PPA-I-catalyzed hydrolysis of
G -pNP in the absence and presence of the inhibitors. The plots
2
Temperature Dependence of Inhibitory Activities of 5-CQA, CA,
and QA. Figure 4 shows the inhibition of the PPA-catalyzed
hydrolysis of G -pNP by 0.1 mM 5-CQA, 0.3 mM CA, and
given at various inhibitor concentrations gave straight lines. The
slope and intercept on the vertical axis of each line increased with
increasing inhibitor concentration. The lines intersected each
other on a point in the second quadrant, suggesting that the
inhibition modes of 5-CQA and CA against PPA-I are mixed-
type. The kcat and Km values for the PPA-I-catalyzed hydroly-
2
2
5 mM QA at pH 6.9 in the temperature range of 20-40 °C. The
relative activities of PPA-I and PPA-II in the presence of 0.1 mM
-CQA or 0.3 mM CA increased linearly with increasing tem-
perature. The activities of both isozymes were inhibited 80% at
5
2
3
0 °C and 40% at 40 °C by 0.1 mM 5-CQA; and 70% at 20 °C and
0% at 40 °C by 0.3 mM CA, suggesting that the inhibitory
-2 -1
sis of G -pNP were determined to be (4.94 ( 0.23) ꢀ 10
s
and
2
activities of 5-CQA and CA are dependent on the reaction
temperature and that the lower the temperature is, the stronger
their inhibitory activities are. It is interesting to note that the
inhibition of PPA by QA was not dependent on temperature, and
4.48 ( 0.34 mM, respectively. The k and K values in the
cat
m
-2 -1
presence of 100 μM 5-CQA were (1.52 ( 0.21) ꢀ 10
s
and
3.04 ( 0.32 mM, respectively, and those in the presence of 300 μM
-
2
-1
CA were (2.31 ( 0.21) ꢀ 10
s
and 3.04 ( 0.38 mM, res-
0
40% of PPA-I and PPA-II activity was inhibited by 25 mM QA in
pectively. The inhibitor constants, K and K , of 5-CQA and CA
i
i
the temperature range examined. These results suggested that the
inhibitory mechanism of QA might be different from that of
against PPA-I were calculated by eq 2. The respective values were
0.23 ( 0.02 mM and 0.05 ( 0.01 mM for 5-CQA and 1.12 (
0.14 mM and 0.27 ( 0.04 mM for CA.
The inhibition kinetics was examined with PPA-II by 5-CQA
and CA under the same conditionsas those examined with PPA-I.
The results obtained with PPA-II were similar to those with PPA-
I (data not shown), suggesting that the inhibition modes of
5-CQA and CA against PPA-II are mixed-type too. The kcat
5-CQA and CA as well as the results shown above (Figure 3).
Mixed-Type Inhibition of PPA-I and PPA-II by 5-CQA and CA.
As the Michaelis-Menten kinetics was observed in the inhibition
of PPA isozymes by 5-CQA and CA (cf. Figure 3A), the kinetics
was examined more precisely. The initial velocity (v) was mea-
sured at 0.80 μM PPA-I and various concentrations (4-12 mM)

9222 J. Agric. Food Chem., Vol. 57, No. 19, 2009
Narita and Inouye
and Km values for the PPA-II-catalyzed hydrolysis of G -pNP
5-CQA against two isozymes are similar, and those of CA and
those of QA are also similar. In other words, these inhibitors do
not discriminate the structural and functional differences between
the isozymes. The inhibitory activities of 5-CQA, CA, and QA
against PPA at pH 9 were reported using a starch substrate and a
crude PPA preparation (the protein content in which was 53%;
purchased from Fluka, Buchs, Switzerland), and the inhibition
potencies were reported to be in the order of 5-CQA > CA >
QA (25). This result agrees apparently with that obtained in the
present article using PPA isozymes and a small synthetic substrate
2
-2
-1
were determined to be (4.97 ( 0.25) ꢀ 10
s
and 4.63 (
0
.35 mM, respectively. The kcat and K values in the presence
m
-2
-1
of 100 μM 5-CQA were (1.78 ( 0.22) ꢀ 10
s
and 3.12 (
0
.43 mM, respectively, and those in the presence of 300 μM CA
-2
-1
were (2.78 ( 0.20) ꢀ 10
s
and 3.32 ( 0.26 mM, respectively.
0
The K and K of 5-CQA against PPA-II were calculated to be
i
i
0
.21 ( 0.02 mM and 0.05 ( 0.01 mM, respectively, and those of
CA were 1.64 ( 0.30 mM and 0.25 ( 0.03 mM, respectively
(
Table 1).
The mixed-type inhibition suggests that there might be an
G -pNP, and the inhibitory potencies of 5-CQA, CA, and QA
2
inhibitor-binding site (or I-site) for 5-CQA and CA on the enzyme
are likely to be similar to those of starch and G -pNP substrates.
2
(
PPA-I and PPA-II) surface in addition to the substrate-binding
We have examined preliminarily the inhibition of PPA activity by
the inhibitors in starch hydrolysis, although 60% of PPA activity
remained even at excess inhibitor concentrations, such as 1 mM
5-CQA, suggesting that the inhibitory potency of the inhibitors
might be weaker in the PPA-catalyzed hydrolysis of starch
0
site (or S-site). It is noteworthy that the K value is much lower
than the K in all K and K pairs, indicating that both 5-CQA and
i
0
i
i
i
CA bind tighter to the I-site on the substrate-bound form of PPA-
I and PPA-II than that on the free form. It is also noted that the
Km values in the presence of the inhibitors are lower than that in
their absence. In other words, binding of the substrate to the S-site
decreases the binding affinity of the inhibitor to the I-site, whereas
binding of the inhibitor to the I-site increases the binding affinity
of the substrate to the S-site.
than that of G -pNP. We should point out the differences
2
in the assay methods applied for PPA activity for starch and
G -pNP. The reducing-activity measurement was applied to
2
starch hydrolysis (25), but we have observed that it is strongly
affected by carboxylic acids and sugar moieties of the inhibitors,
although this point was not considered in the previous re-
ports (25). Re-evaluation of the inhibitory potencies of 5-CQA,
CA, and QA in the PPA-catalyzed hydrolysis of starch is
currently underway with the development of a new assay method.
Although we have no direct evidence whether the three
inhibitors interact with the same I-site or different ones on the
enzyme surface, we propose a hypothetical model for the I-site on
the basis of the IC50 values (Table 1) by mimicking the subsite
structures demonstrated in the I-sites of proteinases (30-33). In
this model, the three inhibitors bind to the same I-site which is
separated into two subsites, CA and QA subsites. Here, we
introduce a concept that the structure of 5-CQA is separated
into CA and QA substructures, which are linked with an ester
bond to form 5-CQA. CA and the CA substructure in 5-CQA
could bind to the former subsite and QA and the QA substructure
to the latter subsite. The interaction of CA with the CA subsite
increases the inhibitory activity 70 times higher than that of QA
with the QA subsite (Table 1). It was suggested that the CA
substructure rather than the QA one may play a more significant
role in the inhibitory activity of 5-CQA and that the inhibitory
potency of CA is enhanced 5 times by combining with QA to form
0
Temperature Dependence of the Inhibitor Constants (K and K )
i
i
0
of 5-CQA and CA. The van’t Hoff plots of K and K at pH 6.9 at
temperatures of 20, 25, 30, 35, and 40 °C were examined (data not
shown). From the slope of the plots, the standard enthalpy
changes (ΔH ) for the bindings of inhibitors (5-CQA and CA)
with PPA isozymes were determined. The standard Gibbs energy
changes (ΔG ) and entropy changes (ΔS ) at 25 °C were also
calculated from eqs 3 and 4 (Table 2). These results indicated that
the binding of PPA-I or PPA-II with 5-CQA or CA was
i
i
o
o
o
o
accompanied with a large ΔH value, and thus the reaction was
exothermic. It was shown that the binding was driven by a
large increase in enthalpy (enthalpy-driven reaction). Enthalpy-
entropy compensation was noticed in all cases.
DISCUSSION
Inhibitory Activities of 5-CQA, CA, and QA against PPA-I and
PPA-II. The inhibitory activities of 5-CQA, CA, and QA against
PPA-I and PPA-II were assayed with G -pNP substrate at pH 6.9
and 30 °C, and the inhibition potencies against both isozymes
were evaluated to be in the order of 5-CQA > CA . QA by com-
paring the IC50 values (Table 1). The inhibitory potencies of
2
5-CQA. In other words, there might be positive cooperativity
between the interactions of CA and QA substructures with their
respective subsites. It is interesting to note that the inhibitory
potency of a metalloproteinase inhibitor, phosphoramidon
Table 1. Inhibitor Constants and IC50 of 5-CQA, CA, or QA against PPA-I or
PPA-II
a
0
enzyme
inhibitor
IC50 (mM)
K
i
(mM)
K
i
(mM)
[
L
N-(R-
L-rhamnopyranosyl-oxyhydroxyl-phosphophinyl)-L-Leu-
-Trp], against thermolysin is drastically enhanced when the
PPA-I
5-CQA
CA
QA
0.08 ( 0.02
0.40 ( 0.03
26.5 ( 1.8
0.07 ( 0.02
0.37 ( 0.04
25.3 ( 0.7
0.23 ( 0.02
1.12 ( 0.14
NA
0.05 ( 0.01
0.27 ( 0.04
NA
rhamnopyranosyl substructure is removed, suggesting that there
is negative coopreativity between the interaction of the rhamno-
pyranosyl substructure with the S1 subsite of the thermolysin
active site and that of the remaining phosphoramidate substruc-
b
PPA-II
5-CQA
CA
QA
0.21 ( 0.02
1.64 ( 0.30
NA
0.05 ( 0.01
0.25 ( 0.03
NA
0
0
ture with the S1 -S2 subsites (34, 35).
a
b
The inhibitory mode of QA was unique and hardly analyzed
with a simple Michaelis-Menten-type interaction between the
Each value is a mean of triplicate analysis ( standard deviation. NA, samples
not analyzed.
Table 2. Thermodynamic Parameters for the Interactions of 5-CQA or CA with PPA-I or PPA-II (Parameter Unit: kJ mol-1 a
)
o
o
o
o
o
o
enzyme
inhibitor
ΔG Ki
ΔG Ki
0
ΔH Ki
ΔH Ki
0
TΔS Ki
TΔS Ki
0
PPA-I
5-CQA
CA
-21.7 ( 1.7
-17.9 ( 2.5
-22.8 ( 2.3
-16.6 ( 1.7
-25.5 ( 1.9
-18.0 ( 2.6
-25.7 ( 2.5
-22.0 ( 2.1
-70.3 ( 4.3
-78.9 ( 6.2
-67.9 ( 9.3
-83.0 ( 4.4
-59.0 ( 3.6
-58.3 ( 5.9
-59.1 ( 6.1
-69.3 ( 4.1
-48.5 ( 3.9
-61.0 ( 6.0
-45.0 ( 8.9
-66.4 ( 4.2
-33.4 ( 3.7
-40.3 ( 5.8
-33.4 ( 5.9
-47.3 ( 3.9
PPA-II
5-CQA
CA
a
o
o
Each value is a mean of triplicate analysis ( standard deviation. ΔG and TΔS values were given at 25 °C.

Article
J. Agric. Food Chem., Vol. 57, No. 19, 2009 9223
enzyme and inhibitor (Figure 3B). The inhibitory mode of QA
must be examined further in the next research step kinetically and
thermodynamically. Presently, as a matter of convenience, when
it is assumed that PPA and QA interact at the ratio of 1:1 (mol/
mol) and that the IC50 (26.5 mM) of QA against PPA gives an
o
approximate K value, the ΔG for the interaction between PPA
i
-1
and QA was calculated by eq 3 to be -8.9 kJ mol at 25 °C.
The ΔG values for the PPA-I inhibition by 5-CQA and CA are
compared (Table 2). The contribution of CA is 83% in the ΔG
value of 5-CQA, while it is 71% in the ΔG Ki
o
o
Ki
o
0
value, suggesting
that the substrate-binding to the S-site of PPA-I enhances the
binding affinity of 5-CQA more than that of CA. Interestingly,
the contribution of CA in the inhibition of PPA-II is 73% in the
o
Ki
o
ΔG value, while it is 86% in the ΔG Ki value, suggesting that
0
the substrate-binding to the S-site of PPA-II attenuates the
binding affinity of 5-CQA more than that of CA. The inhibition
o
of PPA-I by 5-CQA gives the ΔG
value of -21.7 ( 1.7 kJ
Ki
-
1
-1
o
mol and that by CA gives the value of -17.9 ( 2.5 kJ mol
indicating that the QA substructure in 5-CQA might contribute
to the ΔG value of 5-CQA by ΔΔG (QA) of -3.8 kJ mol
The hypothetical contribution [ΔΔG (QA) or ΔΔG
,
Figure 6. Relationship between the enthalpic changes (ΔH ) and en-
o
tropicchanges(TΔS ) fortheinteractionsofthePPAisozymes(PPA-Iand
o
Ki
o
-1
.
PPA-II) with 5-CQA and CA. The enthalpy-entropy compensation was
Ki
o
o
o
o
0
(QA)]
indicated. The ΔH and TΔS values derived from the K
values are shown
values are with closed ones. The
Ki
Ki
i
0
of the QA substructure in the binding affinity of 5-CQA to PPA
with open symbols, and those from the K
i
o
was estimated likewise. The ΔΔG
0
o
(QA) value for PPA-I is
interactions of PPA-I with 5-CQA and CA are shown with circles and
triangles, respectively, and those of PPA-II with 5-CQA and CA are with
squares and diamonds, respectively. The solid line was drawn by best
fitting to the data. The slope of the line is 0.761 ( 0.070, and the correlation
coefficient R is 0.976.
Ki
-
1
o
-
7.5 kJ mol . For PPA-II, the ΔΔG (QA) and ΔΔG
0
(QA)
Ki
Ki
-
1
are -6.2 and -3.7 kJ mol , respectively. These results suggest
that the QA substructure contributes more strongly to the
interaction between 5-CQA and the PPA-I I-site when the S-site
is accommodated with the substrate than when it is vacant, and
inversely, the QA substructure contributes more strongly to the
interaction between 5-CQA and the PPA-II I-site when the S-site
is vacant than when it is accommodated with the substrate
inhibited by 2 mM 5-CQA and by 4 mM CA (Figure 3A),
indicating that the ESI complexes of the isozymes formed with
0
5-CQA or CA should not be active, or k þ2 , kþ2, although these
(
36, 37).
The ΔΔG (QA) and ΔΔG
rate constants were not determined, and the formation of the ESI
complex as well as EI complex was not evidenced. These points
will be investigated more precisely by rapid kinetics with the
stopped-flow method and X-ray crystallographic analysis in the
next step.
o
Ki
o
0
(QA) values, which are con-
Ki
sidered to be measures of the contribution of the QA substructure
in the interaction between 5-CQA and PPA isozymes, are in the
-1
range of (-3.7)-(-7.5) kJ mol (32,33). Although these values
o
-1
are lower than the ΔG value of -8.9 kJ mol estimated from the
PPA inhibition by QA, it could be safely stated that the binding
affinity of 5-CQA to PPA was given roughly by the addition of
those of CA and QA. This result supports our hypothetical model
given above that the 5-CQA binding siteonthe PPA-I and PPA-II
isozymes is composed of two subsites which accommodate the
CA and QA substructures of 5-CQA, separately. We are currently
examining the interaction between the inhibitors and PPA
isozymes and also characterizing the I-site by fluoro-spectro-
scopic and surface-plasmon resonance spectroscopic analyses
and chemical modification of the amino acid residues of PPA.
The inhibitory activity of the CA plus QA mixture at various
ratios and that of other CQA species such as 3-CQA, 4-CQA,
Thermodynamic Parameters for the Interactions of 5-CQA and
o
CA with PPA-I and PPA-II. The ΔH value was determined from
the slope of the van’t Hoff plots in the temperature range of
o
o
20-40 °C. The ΔG and ΔS values at 25 °C were calculated using
eqs 3 and 4 (Table 2). These results indicated that the interaction
of PPA-I or PPA-II with 5-CQA or CA was exothermic and gave
o
a large increasein ΔH , indicatingthat the interaction is enthalpy-
driven. The plots were fitted to the regression line with a slope of
0.761 ( 0.070 (Figure 6). This value is close to the value of 1, and
thus, the enthalpy-entropy compensation is noticed in all inter-
actions of PPA isozymes with 5-CQA or CA. This type of
compensation appears to be a widespread phenomenon and is
widely invoked as an explanatory principle in thermodynamic
analyses of proteins, ligands, and so forth. It has been suggested
that this compensation is an intrinsic property of complex,
fluctuating, or aqueous systems. When PPA and inhibitors
interact to form EI complexes, the states of solvation of the
enzyme and inhibitors might be changed accompanied with large
changes in enthalpies and entropies, and thus with very little effect
on the Gibbs energies of solvation (38-40).
3
,4-di-CQA, 3,5-diCQA, 3,4,5-triCQA, and 5-feruloyl-QA will be
examined to further characterize the subsites for accommodating
the CA and QA substructures.
Inhibititory Models of 5-CQA and CA against PPA-I and PPA-
II. The dependence of Δv on the inhibitor (5-CQA and CA)
concentration [I] shows a standard profile of the Michaelis-
Menten-type kinetics. In addition, the linear relationship between
[
I]/Δv and [I] suggests that the enzyme (PPA-I and PPA-II) binds
Inhibitor Constants of 5-CQA and CA against PPA-I or PPA-II.
0
to each inhibitor (5-CQA or CA) at a molar ratio of 1:1. The
inhibitory modes in all cases, namely, inhibition of PPA-I and
PPA-II by 5-CQA and that by CA, were shown to be mixed-type
inhibition (cf. Figure 5). In the general model for mixed-type
inhibition (29, 36, 37), the ternary ESI complex formed with the
The inhibitor constants K
dissociation constants K
i
and K
i
that correspond to the
of the EI complex into I plus E and
d
that of the ESI complex into I plus ES complex were determined
from eq 2 (Table 1). It was indicated that both 5-CQA and CA
0
showed mixed-type inhibition with K
> K
, suggesting that the
i
i
enzyme (E), inhibitor (I), and substrate (S) is enzymatically active
inhibitors bind to the ES complex stronger than to the enzyme.
0
0
with a molecular activity of k , while the ES complex has that of
The inhibitor constants K
i
and K
i
of acarbose, an antidiabetic
þ2
k
þ2
. In the present study, both PPA isozymes were completely
drug, against PPA in the hydrolysis of maltopentaose at pH 6.9

9224 J. Agric. Food Chem., Vol. 57, No. 19, 2009
Narita and Inouye
and 30 °C were reported to be 3 μM and 0.62 μM, respec-
(15) Pasero, L.; Mazzei-Pierron, Y.; Abadie, B.; Chicheportiche, Y.;
Marchis-Mouren, G. Complete amino acid sequence and location
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chim. Biophys. Acta 1986, 869, 147–157.
0
i
tively (41). The K and K values of 5-CQA against PPA-I in the
i
hydrolysis of G -pNP are 0.23 ( 0.02 mM and 0.05 ( 0.01 mM,
2
respectively. Acarbose is a potent R-amylase inhibitor, and its
(
(
(
16) Marchis-Mouren, G.; Pasero, L. Isolation of two amylases in porcine
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inhibitory activity is 100 times stronger than that of 5-CQA.
5-CQA may not be applicable for therapeutic use in humans
because of its lower inhibitory activity in comparison with that
of acarbose, but ingestion of 5-CQA from processed foods
and beverages may be useful for the prevention of diabetes and
obesity and for the management of borderline diabetes in
patients.
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ACKNOWLEDGMENT
We thank Dr. Seunjae Lee (Kyoto University and presently
Samsung Everland, Ltd., Seoul) and R. Kimura and T. Fukunaga
(
20) Sakano, Y.; Takahashi, S.; Kobayashi, T. Purification and proper-
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White bean amylase inhibitor administered orally reduces glycemia
in type 2 diabetic rats. Br. J. Nutr. 2006, 96, 539–544.
(UCC Ueshima Coffee Co.) for their valuable advice and fruitful
comments.
(
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