On the mechanism of a-amylaseAcarbose and cyclodextrin inhibition of barley amylase isozymes Naı¨ma Oudjeriouat1, Yann Moreau2, Marius Santimone1, Birte Svensson3, Guy Marchis-Mouren1 an
Trang 1On the mechanism of a-amylase
Acarbose and cyclodextrin inhibition of barley amylase isozymes
Naı¨ma Oudjeriouat1, Yann Moreau2, Marius Santimone1, Birte Svensson3, Guy Marchis-Mouren1
and Ve´ronique Desseaux1
1
IMRN, Institut Me´diterrane´en de Recherche en Nutrition, Faculte´ des Sciences et Techniques de St Je´rome, Universite´
d’Aix-Marseille, France;2IRD, Institut de Recherche pour le De´veloppement, UR081 Gamet c/o CEMAGREF Montpellier, France;3Carlsberg Laboratory, Department of Chemistry, Copenhagen Valby, Denmark
Two inhibitors, acarbose and cyclodextrins (CD), were
used to investigate the active site structure and function
of barley a-amylase isozymes, AMY1 and AMY2 The
hydrolysis of DP 4900-amylose, reduced (r)
DP18-malto-dextrin and maltoheptaose (catalysed by AMY1 and
AMY2) was followed in the absence and in the presence of
inhibitor Without inhibitor, the highest activity was
obtained with amylose, kcat/Km decreased 103-fold using
rDP18-maltodextrin and 105 to 106-fold using
maltohep-taose as substrate Acarbose is an uncompetitive inhibitor
with inhibition constant (L1i) for amylose and maltodextrin
in the micromolar range Acarbose did not bind to the
active site of the enzyme, but to a secondary site to give an
abortive ESI complex Only AMY2 has a second
secon-dary binding site corresponding to an ESI2 complex In
contrast, acarbose is a mixed noncompetitive inhibitor of
maltoheptaose hydrolysis Consequently, in the presence of
this oligosaccharide substrate, acarbose bound both to the active site and to a secondary binding site a-CD inhibited the AMY1 and AMY2 catalysed hydrolysis of amylose, but was a very weak inhibitor compared to acarbose b- and c-CD are not inhibitors These results are different from those obtained previously with PPA However in AMY1, as already shown for amylases of animal and bacterial origin, in addition to the active site, one secon-dary carbohydrate binding site (s1) was necessary for activity whereas two secondary sites (s1 and s2) were required for the AMY2 activity The first secondary site in both AMY1 and AMY2 was only functional when sub-strate was bound in the active site This appears to be a general feature of the a-amylase family
Keywords: amylose; maltodextrin; acarbose; barley a-amy-lase; binding site
a-Amylase is a retaining glycoside hydrolase of family 13
acting on a-1,4 internal glycoside linkages in starch and
related sugars [1] a-Amylases occur widely in higher plants,
animals, bacteria and fungi and are applied in several
important industries, e.g in starch processing, paper
treatment, pharmaceutical and the food manufacturing
[2–4] Cereal a-amylases, such as barley isozymes AMY1
and AMY2, play an essential role during seed germination
(malting) by hydrolysing the storage starch granules present
in the endosperm AMY1 and AMY2 have 80% sequence
identity [5,6] AMY1 was more active toward starch
granules and more stable at low pH, while AMY2, the
major isozyme, was more active toward nitrophenylated maltooligosaccharides and was inhibited by the proteina-ceous barley a-amylase/subtilisin inhibitor (BASI) to which AMY1 is insensitive [7,8]
Subsite mapping showed that the substrate binding cleft
of both isozymes contains 10 consecutive subsites recogni-zing substrate glucose residue, i.e six toward the nonreduc-ing end and four toward the reducnonreduc-ing end relative to the bond to be cleaved [9] The AMY1 and AMY2 active sites are twice as long as that of the human and porcine enzymes containing only five subsites [9–12] In addition, a noncata-lytic site that facilitated adsorption onto starch granules (and most probably also hydrolysis of starch granules) has been found in barley a-amylase [7,13,14] Binding of b-cyclodextrin at this site inhibits the a-amylase catalysed hydrolysis of starch granules, but no inhibition was observed with soluble substrate [15,16] In AMY2, differ-ential labelling of tryptophan residues using b-CD for protection identified Trp276-Trp277 in this binding site [13] Trp206 belongs to the active site where it is situated at subsite +2 [14] Known crystal structures of a-amylases contain a central catalytic (b/a)8barrel domain (domain A) having an irregularly structured small domain B protruding between b-strand 3 and a-helix 3 of the barrel, and a C-terminal, domain C, folded as an antiparallel b-sheet [17–23] Acarbose is a pseudotetrasaccharide inhibitor of a-amylase, that acts like a transition-state analogue [7] and
Correspondence to V Desseaux, IMRN case 342, Faculte´ des Sciences
et Techniques, Avenue Esc Normandie-Niemen, 13397 Marseille
cedex 20, France Fax: + 33 4 91 28 84 40,
E-mail: veronique.desseaux@univ.u-3mr.fr
Abbreviations: AMY, barley a-amylase; AMY1, barley a-amylase
isozyme 1; AMY2, barley a-amylase isozyme 2; PPA, porcine
pan-creatic a-amylase; CD, cyclodextrin; DP, degree of polymerization;
rDP18, reduced DP18-maltodextrin; G7, maltoheptaose.
Enzyme: a-amylase [a(1,4)-glucan-4-glucanohydrolase; EC 3.2.1.1].
Note: This paper is dedicated to the late Prof E Prodanov
(Montevideo, Uruguay).
(Received 17 March 2003, revised 23 May 2003,
accepted 30 June 2003)
Trang 2binds to the active site [2,14,17] The crystallography of
AMY2/acarbose showed that both the active site,
contain-ing Trp206, and the secondary so-called starch granule
binding site at the surface, containing Trp276-Trp277, bind
acarbose [14] This surface binding site revealed a
charac-teristic stacking of a disaccharide unit from acarbose onto
the Trp residues [14] The starch binding site was required
when acting on insoluble substrates such as starch granules
Previously, acarbose was demonstrated to be a mixed
noncompetitive-type inhibitor of the hydrolysis of amylose,
rDP18-maltodextrin and maltopentaose catalysed by
por-cine pancreatic [24–28] and human [29] a-amylases
Depending on the substrate, one or two secondary
carbo-hydrate binding site(s) were found which became functional
upon substrate binding These sites may be involved in the
catalytic process and/or in product release [24] The same
inhibition type using amylose as substrate was also reported
using amylases from a fish (Tilapia) [30] and a bacterium
(Lactobacillus) [31] The a-amylase mechanism for
hydro-lysis of soluble substrates includes several steps (a) internal
binding to the amylose chain (b) splitting of the chain (c)
and according to the multiple attack hypothesis [32] further
hydrolysis near the reducing end of the nonreducing moiety
of the initially cleaved amylose to liberate successively 1, 2,
3, etc molecules of maltose or longer oligosaccharide(s)
Secondary binding sites are probably required in such a
mechanism for binding and sliding of the substrate chain
Barley AMY1 and AMY2 have a degree of multiple attack
toward amylose of two (B Kramhøft and B Svensson,
unpublished data)
The goal of the present work is to characterize further
the AMY1 and AMY2 function toward soluble
sub-strates The kinetics of hydrolysis of substrates of different
length: i.e DP 4900-amylose, rDP18-maltodextrin and
maltoheptaose, in the presence and in the absence of the
inhibitor acarbose, respectively, of the potential inhibitors
a-, b- and c-cyclodextrin are reported Using a statistical
analysis of the data, the inhibitory mechanism is
investi-gated Moreover the present results are compared with
those obtained recently in our laboratory using amylases
from different species (porcine [24–28], human [29],
Tilapia [30] and Lactobacillus [31]) The inhibitor and
the inhibition type characterize the active site of the
different enzymes and the secondary site(s) needed for
soluble substrate(s) which appear(s) to be a general feature
of a-amylases
Materials and methods
Materials
Barley a-amylases, AMY1 and AMY2, were purified from
green and kilned malt, respectively, according to Svensson
et al [33] and Ajandouz et al [9] Purified AMY1 and
AMY2 gave single bands in SDS/PAGE (not shown)
in amounts corresponding to approximately 5 and
100 mgÆL)1 The amylase concentrations were determined
by measuring A280 (A1%
280 ¼ 24) [24] Amylose (type III from potato) DP 4900 (794 kDa) [34], maltoheptaose,
maltohexaose, maltopentaose, maltotetraose, maltotriose,
maltose, glucose and neocuproin hydrochloride were from
Sigma Maltodextrin of average DP18 (2.9 kDa) was
from Hayashibara Biochemical Laboratories (Okayama, Japan) Reduction of the DP18-maltodextrin to the corresponding alcohol was performed as earlier described [35] by using NaBH4 This was done to facilitate the reducing sugar assay by minimizing the contribution from the substrate to achieve low blank values Acarbose (O-4,6-dideoxy-4-{[4,5,6-trihydroxy-3-hydroxymethyl-2-cyclo-hexen-1-yl]amino}-a-D-glucopyranosyl-(1fi 4)-O-a-D -gluco-pranosyl-(1fi 4)-D-glucose) was generously supplied by Bayer Pharma (France) a-, b- and c-cyclodextrins were from Sigma
Kinetics Kinetic experiments were performed at 30C in 20 mM
sodium acetate buffer (pH 5.5) containing 1 mM CaCl2
and 1 mM sodium azide Substrate, inhibitor and buffer were mixed and the reaction was initiated by adding the enzyme
When amylose or rDP18-maltodextrin was the substrate, the incubation volume was 400 lL and the enzyme volume
100 lL More than 10 concentrations of the substrates, amylose (0.003–0.32 gÆL)1 or 0.038–0.4 lM for AMY1; 0.048–0.8 gÆL)1 or 0.06–1 lM for AMY2) and rDP18-maltodextrin (0.06–1.46 gÆL)1 or 20–500 lM for both isozymes) were used The final concentration of AMY1 and AMY2 was 2.0 nMand 1.0 nM, respectively Acarbose was used in the range 10–80 lMand a-, b- and c-CD were
in the ranges 2–20 mM, 3.2–24 mM, and 1.6–13.6 mM, respectively The reaction was stopped at appropriate time intervals (1, 3 and 5 min) by adding 500 lL of chilled 0.38Msodium carbonate containing 1.8 mMcupric sulfate and 0.2Mglycine (500 lL) and kept on ice [36] The rate
of hydrolysis of amylose and rDP18-maltodextrin was obtained from the increase in reducing power and using maltose as standard
When maltoheptaose was the substrate, the incubation volume was 900 lL and the enzyme volume 100 lL giving a final concentration of 100 nM More than 10 concentrations
of maltoheptaose (0.15–5 mM) were used Acarbose was in the range 0.75–5 mM Samples (100 lL) were removed at appropriate time intervals (0, 0.15, 0.30, 0.45 and 1.00 min), added to 0.1M NaOH (300 lL) to stop the reaction, and kept on ice until analysis The rate of hydrolysis was determined by measuring the produced maltooligosaccha-rides by high-performance anion-exchange chromatogra-phy (HPAEC) on a Carbopac PA-100 (4 mm· 250 mm) column with elution by a 5–500 mMsodium acetate linear gradient over 20 min in 100 mMNaOH, at a flow rate of 1.0 mLÆmin)1 Detection of oligosaccharide and glucose in the eluate was performed by pulsed amperometric detection (PAD) using the Dionex DX-500 chromatograph as reported previously [25] For quantification glucose, malt-ose, maltotrimalt-ose, maltotetramalt-ose, maltopentamalt-ose, maltohexa-ose and maltoheptamaltohexa-ose were used as standards Values from either reductometry or HPAEC-PAD gave initial velocities
as calculated from the slopes obtained by linear regression
of the linear part of the progress curves, which in turn gave the number of glycoside bonds hydrolysed per minute or the amount of product (glucose and maltohexaose) released per minute, respectively The experiments were repeated three
or four times
Trang 3Statistical analyses of kinetics experiments
Statistical analyses were performed using either the REG,
NLIN, or GLM procedure from the SAS/STAT software
package (Sas Institute Inc, Cary, NC, USA) [37] A
significance level of 0.05 was used in all statistical tests
The initial velocity was measured at fixed inhibitor and
varying substrate concentration In order to determine the
type of inhibition, the kinetic data were analysed using a
general initial velocity equation As discussed earlier
[9,27,28], Eqn (1) applies for the present type of data:
Kmð1 þ1
K li½I þ 1
K li K 2i½I2Þ þ ½Sð1 þ1
L li½I þ 1
L li L 2i½I2Þ ð1Þ
In this equation v is the initial velocity, [E]0 the enzyme
concentration, [S] the substrate concentration, [I] the
inhibitor concentration, Km the Michaelis constant and
K1i, K2i, L1i, L2ithe dissociation constants of the different
abortive complexes, EI, EI2, ESI and ESI2, respectively, as
shown in the scheme below It should be noticed that this
equation applies at steady state and at rapid equilibrium
except in the case of noncompetitive inhibition with a
random mechanism at steady state [24]
Equation (1) corresponds to the following reaction
scheme, where Q and P are the products:
A nonlinear statistical analysis was used Equation (1) was
modified by using the association constants K¢1i, K¢2i, L¢1i
and L¢2i which are the inverse of the corresponding
dissociation constants In this equation, it was easier for a
calculated constant to compare its value relative to zero
rather than to obtain a large value When the association
constant value was close to zero, this meant that the
corresponding abortive complex was not present in
significant amounts Actually, one will use the simplest
equation which best matched the data and the actual
inhibition type
Difference spectroscopy
Difference spectra were determined using a double-beam
Shimadzu UV-2401PC spectrophotometer
Double-com-partment cells (each 0.44 cm light path, 230-QS, from
Hellma) were used for both control cell and sample cell The
cells were thermostated at 30C First, both cells were filled
with 20 mM sodium acetate buffer (pH 5.5) containing
1 mMCaCl2and 1 mMsodium azide to define the baseline
Second, AMY1 (40 lM) was introduced into one
compart-ment of the control and one compartcompart-ment of the sample cell
and the reference line was determined (A0) Then acarbose
(1.7–6.5 mM) was added to the buffer compartment of the
control and to the compartment containing AMY1 in the
sample cell The AMY1 concentration in the control cell
was adjusted accordingly by addition of buffer Spectra
were recorded in the 230–320 nm region at a rate of 0.2 nmÆs)1
Results
Determination of kinetic parameters with substrates
of different sizes The AMY catalysed hydrolysis of DP 4900-amylose, rDP18-maltodextrin and maltoheptaose was first measured
in the absence of inhibitor Statistical analysis of the experimental initial rates (v) was performed using the general Michaelis–Menten initial velocity equation for determination of kcatand Kmand calculation of the catalytic efficiency, kcat/Km The kinetic parameters of AMY1 and AMY2 (Table 1) were rather similar, but depended import-antly on the substrate With amylose and rDP18-maltodex-trin as substrates, under saturating conditions, no difference was observed between kcat of AMY1 and AMY2 for amylose or for rDP18-maltodextrin (Table 1) In contrast, however, for maltoheptaose, AMY2 had three times higher
kcat than AMY1 The Km values were increasing with decreasing substrate length from around 0.2 lM for amy-lose, to around 215 lM for maltoheptaose AMY1 and AMY2 (kcat/Km) were 700 to 1000-fold more active toward amylose than rDP18-maltodextrin, which in turn was 170
to 690-fold superior as substrate than maltoheptaose (Table 1) Thus the longer the substrate, the higher was the activity
Inhibition by acarbose Inhibition of amylose hydrolysis occurred in the presence
of 10–80 lM acarbose and the association constants K¢1i, K¢2i, L¢1i and L¢2i were determined according to the general equation (see Materials and methods) For both AMY1 and AMY2, the association constants K¢1i, K¢2i and L¢2iwere close to zero and could not be determined under these conditions while, L¢1i¼ (62 ± 4)103
M )1and L¢1i¼ (28 ± 3)103M )1, respectively The dissociation constants, calculated from the respective association constants in the corresponding equation (K1i, K2i, L1i and L2i), were given in Table 2 When the association constant values were close to zero, the significant values of the dissociation constants K1i, K2i, and L2i could not be obtained (NS) The closest match to the experimental data corresponded to Eqn (2)
Table 1 The enzyme kinetic parameters of hydrolysis of different sub-strates by barley a-amylase isozymes AMY1 and AMY2 Parameter values are given as ± SEM.
Substrate Enzyme
k cat
(s)1)
K m
(l M )
k cat /K m
(s)1Æ M )1 )
Amylose AMY1 206 ± 12 0.21 ± 0.03 1.0 · 10 9
AMY2 202 ± 10 0.16 ± 0.02 1.3 · 10 9
Maltodextrin AMY1 129 ± 5 79.3 ± 9.8 1.6 · 10 6
AMY2 125 ± 4 71.4 ± 7.0 1.8 · 10 6
Maltoheptaose AMY1 2.02 ± 0.10 213 ± 46 9.5 · 10 3
AMY2 5.62 ± 0.5 217 ± 43 26 · 10 3
Trang 4v=½E0 ¼ kcat½S
Kmþ ½Sð1 þ L0
Eqn (2) represents the following reaction scheme of
uncompetitive inhibition, in which the dissociation constant
has been indicated:
This model included only one abortive complex ESI (I
bound at a secondary site s1) and no significant amount of
acarbose was bound to E as in an ES complex The
reciprocal plot drawn for AMY1 according to Eqn (2)
illustrates this model: parallel straight lines intersect the
ordinate axis as expected, the intercept increasing with
increasing acarbose concentration (Fig 1) A similar plot
was obtained for AMY2 (not shown)
Inhibition of the rDP18-maltodextrin hydrolysis occurred
also in the presence of 10–80 lMacarbose For both AMY1
and AMY2 the association constants K¢1i and K¢2i were
close to zero and L¢1i¼ (67 ± 9) 103M )1, L¢1i¼ (42 ±7)
103
M )1, respectively For AMY1, L¢ value was also close
to zero In this case Eqn (2) applies For AMY2, L¢2i¼ (11 ± 5) 103
M )1and in this case, Eqn (3) accounted for the data With both enzymes, the inhibition was as above) the uncompetitive type The resulting dissociation constants are given in Table 2
v=½E0 ¼ kcat½S
Kmþ ½Sð1 þ L0
1i½I þ L0 1iL0 2i½I2Þ ð3Þ The corresponding reaction scheme is:
indicating no EI complex in significant amount but two complexes, ESI (I bound at s1) and ESI2(I bound at s1and
s2), to be present The inhibition is still uncompetitive and the plot drawn with AMY1 illustrates this model: parallel straight lines intersected the ordinate (Fig 2) A similar plot was obtained for AMY2 (not shown) To summarize, rDP18-maltodextrin hydrolysis by both AMY1 and AMY2 was uncompetitively inhibited by acarbose For AMY1, however, only the ESI inhibition complex was present, while with AMY2 both ESI and ESI2were formed
In contrast, when maltoheptaose is the substrate in the presence of 0.75–5 mM acarbose, the experimental data most closely matched Eqn (4):
v=½E0¼ kcat½S
Kmð1 þ K0
1i½IÞ þ ½Sð1 þ L0
1i½IÞ ð4Þ For AMY1, calculation gave the association constants K¢1i¼ (5.2 ± 1.4) 103
M )1 and L¢1i¼ (0.25 ± 0.09) 103
M )1, K¢2i and L¢2i were close to zero Using AMY2, K¢1i¼ (1.2 ± 0.3) 103M )1and L¢1i¼ (1 ± 0.26) 103M )1, K¢2i and L¢2i were also close to zero The dissociation constant values of K1iand L1iare shown in Table 2 This inhibition was of the mixed noncompetitive type for both isozymes and followed the reaction scheme:
Fig 1 Lineweaver–Burk plots AMY1 with varying amylose and fixed
acarbose concentration [I] as indicated This plot was calculated by
statistical analyses of initial rates of hydrolysis using Eqn (2)
Gra-phical analysis was not possible with our data For this reason no
experimental points are reported The plot is drawn from the
corres-ponding rate equation determined by statistical analysis.
Fig 2 Lineweaver–Burk plots AMY1 with varying rDP18-malto-dextrin concentration and fixed acarbose concentration [I] as indicated This plot was calculated by statistical analyses of initial rates using Eqn (2) Graphical analysis was not possible with our data For this reason no experimental points are reported The plot is drawn from the corresponding rate equation determined by statistical analysis.
Table 2 The inhibition constants and type of inhibition by acarbose for
AMY1 and AMY2 acting on different substrates K 1i , K 2i , L 1i and L 2i
are the EI, EI 2 , ESI and ESI 2 related dissociation constants NS, not
significant values.
Substrate Enzyme
K 1i
(l M )
K 2i
(l M )
L 1i
(l M )
L 2i
(l M )
Inhibition type
Amylose AMY1 NS NS 16 NS Uncompetitive
AMY2 NS NS 36 NS
Maltodextrin AMY1 NS NS 15 NS
AMY2 NS NS 24 95
Maltoheptaose AMY1 194 NS 4 10 3 NS Mixed
AMY2 833 NS 1 10 3 NS Noncompetitive
Trang 5Two abortive complexes were present: EI (I bound at the
active site) and ESI (I bound at a secondary site s1)
The reciprocal plot using estimated values drawn for
AMY1 from Eqn (4) illustrated this model: straight lines
intersected in the 2nd quadrant (Fig 3) at a point close to,
but distinct from the origin (see insert) A similar plot was
obtained with AMY2 (not shown) This apparent
discrep-ancy from the inhibition of the long chain substrate
hydrolysis was associated with the weak affinity of
malto-heptaose for the active site as well as an effect also of the
high acarbose concentrations used
Inhibition by cyclodextrins
In the presence of a-, b- or c-cyclodextrin (2–20 mM, 3.2–
24 mMand 1.6–13.6 mM, respectively) and using DP-4900
amylose, as substrate inhibition of AMY1 and AMY2 only
occurred with a-CD which was a very poor inhibitor
compared to acarbose The inhibition constants (not given)
were in the 10–100 millimolar range No other substrates
were investigated with the cyclodextrins Inhibition of starch
granule hydrolysis by b-cyclodextrin has previously been
reported, however, in agreement with our result on amylose,
soluble starch hydrolysis was not inhibited [15]
Difference spectra of acarbose binding
The inhibition of the amylolytic activity of AMY1 by
acarbose involved, as shown above, specific interactions at
either the active site, as in EI, and/or at the secondary
binding site The binding of acarbose to AMY1 was also
monitored by UV difference spectroscopy A complete
AMY2 study, however, has not been performed The
absorbance difference spectra of AMY1 produced by 1.7–
6.5 mM acarbose showed a major peak at 294–295 nm,
except for the d spectrum (Fig 4A) which for unknown reasons was slightly shifted toward a shorter wavelength These spectra indicated that binding of acarbose perturbed
at least one tryptophan residue [38,39] The size of the peak increased with increasing acarbose concentration and was stable for up to 30 min A shift at 294 nm occurred at longer incubation times and analysis by HPAEC of acarbose-AMY1 mixtures from the sample cell indicated that slow hydrolysis of acarbose took place This showed that acarbose was bound at the active site The UV difference spectra therefore were recorded within less than 30 min after mixing The reciprocal of the normalized absorbance difference [E]0/DA ([E]0¼ AMY1 initial concentration;
DA¼ A) A0) measured at 294 nm was plotted against 1/[I]0([I]0¼ acarboseinitialconcentration)yieldingastraight line (Fig 4B) indicating that one molecule of acarbose (I) binds to one molecule of AMY1 (E) to form the monitored AMY1-acarbose complex (EI) according to the reaction:
as a consequence, the following equation applies:
½E0
DA ¼ Kd
De 1
½I þ
1
Fig 4 UV difference spectroscopy of AMY1 with acarbose (A) Scans from 270 to 320 nm are shown The acarbose concentration (in m M ) was 0.00 (a), 1.70 (b), 2.70 (c), 4.60 (d), 6.5 (e) The AMY1 concen-tration [E] 0 was 38.8 l M decreasing to 37.3 l M by addition of acar-bose A 0 is the AMY1 absorbance without acarbose, A is the absorbance measured at the above acarbose concentrations (B) Reciprocal plot of the difference spectra [E] 0 /(A ) A 0 ) vs 1/[I] 0
(acarbose initial concentration) measured at 294 nm upon adding acarbose to AMY1.
Fig 3 Lineweaver-Burk plots AMY1 with varying maltoheptaose
and fixed acarbose concentration [I] as indicated This plot was
cal-culated by statistical analyses of initial rates using Eqn (4) The insert
enlarges the origin region Graphical analysis was not possible with our
data For this reason no experimental points are reported The plot is
drawn from the corresponding rate equation determined by statistical
analysis.
Trang 6in which DA is the absorbance difference, De is the
difference between the molar absorption coefficients of
the inhibitor complex and the free enzyme, and Kdis the
dissociation constant of the EI complex Equation (5) is of
first order with respect to 1/[I]0and therefore fits a linear
plot It should be noted that Eqn (5) applies only when the
concentration of the inhibitor, I, is much higher than that of
the enzyme, which was the case in the present experiment
Moreover, if more than one molecule of inhibitor binds to
the enzyme and perturbed the spectrum, then the resulting
plot [E]0/DA vs 1/[I]0will not be linear [27] Equation (5)
and Fig 4B were used to determine the dissociation
constant for EI to Kd¼ 0.6 mM which confirmed a
previous determination of the binding constant to AMY1
[7] However, in the light of the present data our
interpret-ation of the data was somewhat different It appeared that
the EI complex was observed by difference spectroscopy
when the concentration [I] was very high when compared to
inhibitor concentrations used in the kinetics studies The
binding of inhibitor at the active center was supported by
the fact that acarbose was slowly hydrolysed to release
glucose in a reaction that followed linear kinetics (not
shown) The question then arose, why the two sites, the
active site and the surface site found by kinetic analysis,
were not both revealed by the difference spectroscopy
Discussion
As shown from the kinetic results obtained in the absence of
inhibitor, amylose was by far the best substrate of barley
amylase Actually, rDP18-maltodextrin was hydrolysed at a
103-fold lower rate and maltoheptaose at 105)106lower rate
than DP 4900-amylose AMY2 was only slightly more
active than AMY1 This finding agreed with the generally
accepted feature that a-amylases are mostly active on long
chain substrate The poor activity of AMY1 and AMY2,
relatively speaking, using maltoheptaose as a substrate is
due, on the one hand to the fact that maltoheptaose at most
occupied 7/10 subsites of the active site in productive
complexes and on the other hand because nonproductive
complexes would inhibit barley a-amylase catalysed
malto-heptaose hydrolysis [9]
When discussing the kinetic results obtained in the
presence of inhibitors, the main question to be asked is
why acarbose apparently did not occupy the active site of
AMY1 and AMY2 when the substrates used are amylose
and maltodextrin; while in all other situations, as shown by
difference spectra, X-ray crystallography and for PPA,
acarbose was bound at the active site A second point is then
how to explain that EI was formed when maltoheptaose was
the substrate How consistent were these corresponding
data and what was the contribution to the knowledge of the
barley isozymes and to the a-amylase family?
As will be discussed further, amylose and
rDP18-maltodextrin most probably have significantly higher
affinity for the active site of AMY (E) than found for
acarbose When the substrates and the inhibitor compete
for the active site, the high affinity of the substrates
facilitates their binding whereas the inhibitor binding does
not occur Therefore, no significant amount of EI complex
was formed and the acarbose inhibition was
uncompeti-tive The ES complex reacted to give either products or
the abortive ESI complex (I bound at s1) When AMY1 was used with the substrates amylose or rDP18-malto-dextrin, only one acarbose molecule was bound to ES as well as to ESI In the case of rDP18-maltodextrin/AMY2, however, an additional acarbose molecule was bound to give ESI2suggesting that one more sugar binding site (s2) was present on the enzyme surface (Fig 5A) Such a site was found in PPA [24] We suggest that this second surface site reflected a certain structural difference between AMY1 and AMY2 To summarize, we propose on the basis of the above kinetic results, that one secondary binding site (s1) in AMY1 and two (s1and s2) in AMY2 were necessary for enzyme activity It (they) became functional only when S was bound at the active site and were thus quite different from the starch granule binding site earlier characterized in cereal amylases In the uncompetitive model, no inhibitor was present at the active site This, however, did not contradict the X-ray data [14] and the present difference spectra The kinetic results showed that acarbose was a poor inhibitor of AMY1 having a poor affinity for the active site Conse-quently at the inhibitor concentrations [I] used, no EI complex was formed At higher concentrations of acar-bose, as used for the difference spectroscopy, the EI complex could form and in accordance with the modest affinity, the dissociation constant was very high (0.6 mM) (Fig 5B) Also, the acarbose concentration (10 mM), used for soaking crystals of AMY2 to get the acarbose/AMY2 complex, was very high [14]
In contrast to the uncompetitive inhibition with amylose and maltodextrin, the inhibition with maltoheptaose was of the mixed noncompetitive type Thus, both the EI and ESI complexes were formed (Fig 5A) The noncompetitive acarbose inhibition may result, firstly because maltohepta-ose was a poor substrate for which AMY1 and AMY2 showed, respectively, 105 and 102-fold lower catalytic efficiency than for amylose and maltodextrin With com-petitive binding to enzyme of the substrate and of the inhibitor, the weak affinity of maltoheptaose to enzyme (E) facilitated the binding of acarbose (I) to allow the formation
of the abortive EI complex, the ES and ESI (I in s1) complexes being also formed (Eqn 4 and Fig 5) It can be concluded that the low affinity of both acarbose and maltoheptaose for the active site was associated with noncompetitive inhibition, while uncompetitive inhibition
as a consequence of amylose and maltodextrin binding with high affinity for AMY
a-CD was a weak inhibitor of AMY catalysed amylose hydrolysis b- and c-CD, however, were not inhibitory In contrast a-, b- and c-CD were all inhibitors of PPA, and active at a slightly lower concentration in 0.25–5 mMrange Such difference most likely reflected the different structures
at the active site of PPA [12] and AMY [9]
Two questions arose from the results of difference spectroscopy of acarbose binding: (a) in the observed EI complex, which binding site was then occupied? Our results support that in EI, acarbose occupied the active site as at prolonged incubation acarbose hydrolysis took place This experiment was of major interest as it allowed determination
of the Kd(the dissociation constant) of EI which could not
be obtained by the kinetics approach when amylose or
rDP-18 maltodextrin were used as substrates The K was
Trang 7actually in the same range as the K1i obtained with
maltoheptaose as substrate (0.2 mM); (b) why do we not
observe the secondary binding site demonstrated
kinetic-ally? Two answers may be proposed: either this site was not
functional (accessible) in the absence of substrate, as
postulated in the conclusion, or acarbose did not bind to
a Trp but to a different residue which could not be
monitored by UV difference spectroscopy
As mentioned above, similar studies have been
conduc-ted on PPA The results were strikingly different from
those obtained with barley AMY Acarbose was a
noncompetitive inhibitor for PPA and an uncompetitive
for AMY when long chain substrates were used In that
case, the inhibitory complex ESI was formed with both
enzymes, however, the EI complex was observed only with
PPA This discrepancy was explained by the higher affinity
of acarbose for PPA as indicated by the lower dissociation constant of the acarbose-PPA complex (1.7 lM) [24] The dissociation constant of the acarbose-AMY complex cannot be determined kinetically but was obtained from the difference spectroscopy analysis (Kd¼ 0.6 mM) Such a large difference probably reflects differences of the struc-ture and the energetics profiles of the respective active sites The comparison of AMY and PPA active site showed large differences in the binding affinities of corresponding subsites [9,10,12] The PPA active site, moreover, had five subsites and acarbose can occupy four of these, while the AMY active site had 10 subsites and this crevice was thus far from completely occupied by acarbose, and acarbose apparently binds with lower affinity Acarbose was thus demonstrated to be a useful tool in describing active sites in different a-amylases
Fig 5 Schematic mechanism for the AMY action of acarbose inhibition and binding (A) Kinetics: S ¼ amylose, rDP18-maltodextrin or malto-heptaose with I ¼ acarbose; S ¼ amylose with I ¼ a-CD K 1i , L 1i , L 2i are dissociation constants (B) Difference spectra Kd is the dissociation constant.
Trang 8Barley isozymes AMY1 and AMY2 were thousand-fold
more active toward amylose than toward maltodextrin and
a million-fold more active than toward maltoheptaose
AMY2 was slightly more active than AMY1 AMY1 and
AMY2 were inhibited by acarbose a-CD was a weak
inhibitor and b- and c-CD were not inhibitory This is in
contrast to the high inhibitory toward porcine [24–28] and
human [29] a-amylases Also the inhibitory mechanism by
acarbose of the amylose and maltodextrin hydrolysis was of
a different type in the barley, compared to the human and
porcine enzymes This different behaviour most probably
reflects the individual active site structures Moreover, in
addition to the active site, the presence of one (s1) or two (s1
and s2) secondary carbohydrate binding sites already found
in amylases from other species were demonstrated
Alto-gether three to four carbohydrate binding sites were
postulated: (a) the starch granule binding site [40] [14]; (b)
the active site; (c) and one and sometimes two secondary
site (s) as deduced from the inhibition kinetics ([24] and the
present work) The precise functions of each site are
unknown but remarkably, the inhibition kinetics
demon-strated that they became functional only when E was bound
to S in the ES complex Conformational changes very likely
occurred that couple the function of these sites with that of
the active site The secondary site(s) might be involved in
substrate hydrolysis and/or product release This function
was then clearly distinct from the barley a-amylase binding
onto starch granules, which most probably occurred prior
to hydrolysis of the substrate glycosidic bond
Acknowledgements
We thank Drs E H Ajandouzand R Koukiekolo for stimulating
discussion, C Villard for advice and excellent technical assistance,
B Dwisusilo for his help in the preparation of the illustration, and
S Ehlers for enzyme purification.
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