Study of the kinetic parameters for synthesis and hydrolysis of pharmacologically active salicin isomer catalyzed by baker’s yeast maltase

Article abstract of DOI:10.1134/S0036024411130346

One of the key elements for understanding enzyme reactions is determination of its kinetic parameters. Since transglucosylation is kinetically controlled reaction, besides the reaction of synthesis, very important is the reaction of enzymatic hydrolysis of created product. Therefore, in this study, kinetic parameters for synthesis and secondary hydrolysis of pharmacologically active α isosalicin by baker’s yeast maltase were calculated, and it was shown that specifity of maltase for hydrolysis is approximately 150 times higher then for synthesis.

Full text of DOI:10.1134/S0036024411130346

ISSN 0036ꢀ0244, Russian Journal of Physical Chemistry A, 2011, Vol. 85, No. 13, pp. 2317–2321. © Pleiades Publishing, Ltd., 2011.
CHEMICAL KINETICS
AND CATALYSIS
Study of the Kinetic Parameters for Synthesis and Hydrolysis
of Pharmacologically Active Salicin Isomer Catalyzed
by Baker’s Yeast Maltase1
D. V. Veli kovica, A. S. Dimitrijevicb, F. J. Bihelovicb, R. M. Jankovb, and N. Milosavicb
c
a Innovation Center of the Faculty of Chemistry, University of Belgrade, Studentski trg 12, Belgrade, Serbia
b Faculty of Chemistry, University of Belgrade, Studentski trg 12, Belgrade, Serbia
eꢀmail: nenadmil@chem.bg.ac.rs
Received December 30, 2010
Abstract—One of the key elements for understanding enzyme reactions is determination of its kinetic
parameters. Since transglucosylation is kinetically controlled reaction, besides the reaction of synthesis, very
important is the reaction of enzymatic hydrolysis of created product. Therefore, in this study, kinetic paramꢀ
eters for synthesis and secondary hydrolysis of pharmacologically active
α isosalicin by baker’s yeast maltase
were calculated, and it was shown that specifity of maltase for hydrolysis is approximately 150 times higher
then for synthesis.
Keywords: synthesis and hydrolysis of salicin isomer, catalysis by maltase, kinetic parameters.
DOI: 10.1134/S0036024411130346
INTRODUCTION
noside] and its structural isomers exhibit pronounced
anticoagulant activity [5], tyrosinase inhibitory activꢀ
ity [6], and are also used as antiꢀinflammatory, analgeꢀ
Many glycoside hydrolases exhibit transglycosylaꢀ
tion activity, and transglycosylation reactions are well
known and widely used methods for glycoside syntheꢀ
sis [1]. Transglycosylation is a mechanism for glycoꢀ
sidic bond formation in which a glycosyl moiety from
a donor compound is transferred to the hydroxyl group
of an acceptor molecule other than water [2] (Fig. 1).
Transglucosylation is subtype of transglycosylation,
where enzyme transfers glucose moiety to acceptor
molecule.
sic and antipyretic prodrugs [7, 8]. Moreover,
α
ꢀgluꢀ
coside of orthoꢀhydroxybenzyl alcohol ( ꢀisosalicin)
α
is more potent anticoagulant than naturally occurring
salicin [5].
Alphaꢀglucoside of orthoꢀhydroxybenzyl alcohol,
like all other glycosides, is formed in a kinetically conꢀ
trolled reaction that shows optimum in time, when
disaccharide is used as donor [9]. Because the reaction
is controlled kinetically, it is possible to overshoot the
equilibrium conversion of reactant into product. As
the reactant is consumed the concentration of the
product will reach the peak when rates for its synthesis
and debenzylation become equal. At this point kinetic
control is lost and the reaction should be stopped
before thermodynamic control takes over and the
product undergoes enzymatic hydrolysis (secondary
Principally, all glycosidases can be divided in two
classes, depending whether hydrolysis occurs with
retention or with inversion of configuration at the anoꢀ
meric center. Maltase (EC 3.2.1.20;
α
ꢀglucosidase)
belongs to the family of glycosidases which hydrolyze
substrate and form products with the same configuraꢀ
tion at the anomer atom like substrate [3]. It is one of
the most abounded glycosyl hydrolases present in
baker’s yeast (Saccharomyces cerevisiae), and has been hydrolysis in Fig. 1) [3]. Hence, the yield of glucoside
used for synthesis of various pharmacologically active is determined by a delicate balance between the rates
glycosides [4]. Sometimes, the glycosidic residue is
crucial for glucosides activities; in other cases glycosyꢀ
lation improves pharmacokinetic parameters [5]. We
of [E–G +A] synthesis (K1
K7 2) on the one hand, and product hydrolysis (k6
–5), on the other.
,
k
3) and hydrolysis (k4
,
k–3
,
,
k
,
k5,
k
used maltase for synthesis of
benzylꢀ ꢀDꢀglucopyranoside] with orthoꢀhydroxyꢀ
α
ꢀisosalicin [2ꢀhydroxyꢀ
Determination of kinetic parameters that lies in K1
is earlier described for ( ꢀgalactosidase [2], as well as
reaction driven with K7 for ꢀglucosidase (substrate
transglycosylation) [10], although this reaction does
not have influence in transglucosylation reaction catꢀ
alyzed by maltase from baker’s yeast. Reaction of inhiꢀ
α
β
benzyl alcohol as glucose acceptor and maltose as gluꢀ
cose donor in reaction of transglucosylation (Fig. 1).
Salicin, [2ꢀ(hydroxymethyl)phenylꢀβꢀDꢀglucopyraꢀ
β
1
The article is published in the original.
2317

ꢀ
C
VELI KOVI C et al.
2318
[E+GA]
H2O
k6
k5
kꢁ5
GA
A
H2O, k2
Kig
K7
M−G+[E]
[E+G]
E−G
E
M
G
K1
M
G
kꢁ3
A
k4
k3
E−G+A
Fig. 1. Schematic model for different kind of reactions catalyzed by
αꢀglucosidase (E), with maltose (M) as glucose (G) donor.
The role of acceptor can be played by water (primary hydrolysis ) or by other hydroxylic compounds (transglucosylation) such
k
1
as, orthoꢀhydroxybenzyl alcohol (A), hydrolysis products (G), or a second substrate molecule, i.e. maltose (M), (substrate transꢀ
glucosylation), [E–G] represents covalent complex between enzyme and glucose, and GA is glucoside of orthoꢀhydroxybenzyl
α
alcohol; corresponds secondary hydrolysis.
k
6
bition glycosidase (
described for ꢀgalactosidase [11].
Therefore, in this study a detailed kinetic analysis for
synthesis and hydrolysis of pharmacologically active
alpha glucoside of orthoꢀhydroxybenzyl alcohol by malꢀ
tase was performed, because they are essential kinetic
parameters crucial for maximal yield of reaction.
K
ig) by monosaccharide is also
α
ꢀisosalicin (0.35 mM) as another in simultaneous
β
reaction, in 50 mM phosphate buffer pH 6.8. Concenꢀ
tration of enzyme was 100 U/ml. Blank probe was perꢀ
formed in the same reaction condition only without
α
ꢀisosalicin and with enzyme concentration of
75 U/ml. Concentration of liberated 4ꢀnitrophenolate
ion was monitored in UV/VIS spectrophotometer at
445 nm in kinetic mode.
After the
by monitoring rate of hydrolysis
tase in condition saturated with
The kinetic was monitored in the same HPLC condiꢀ
tion as described in previous section but with 25 U per
mL of reaction mixture.
K
m was obtained,
k
cat has been determined
ꢀisosalicin by malꢀ
ꢀisosalicin (10 m).
EXPERIMENTAL
α
α
K
Enzyme ꢀglucosidase (E.C. 3.2.1.20) was isolated
α
from baker’s yeast by a slightly modified previously
published procedure [12]. All commercially available
reagents and solvents were used as obtained without
further purification.
RESULTS AND DISCUSSION
Determination of kinetic parameters for synthesis of
α
ꢀisosalicin. Glucose donor for this reaction (Fig. 1)
Determination of kinetic parameters for synthesis of
ꢀisosalicin. Applying method of King and Altman
[13] under the conditions of the stadyꢀstate for all
enzyme substrate complexes [10] at Fig. 1, following
kinetic parameters are given as:
was 1.2 M maltose in 0.1 M phosphateꢀcitrate buffer
pH 6.8, and concentration of orthoꢀhydroxybenzyl
alcohol was varied from 0 to 40 mM. The enzymatic
reaction was stopped by adding 0.1 M HCl to give
pH 3.0 and acetonitrile (AN) to give 10% (v/v). The
reaction mixtures were then centrifuged, and analyzed
α
const + (coefGA)[GA] + (coefM)[M]
Km = ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ ,(1)
s
using an Akta Purifier HPLC applying 10
mixture on it. Column used, was Waters Spherisorb
m ODS 2, 4.6 250 mm. Solvent A was 10% aceꢀ
μl reaction
(coefA) + (coefGA ⋅ A)[GA] + (coefM)[M]
where Km represents Michaelis constant for ortho
ꢀ
5
μ
×
s
tonitrile (AN) in 1 mM HCl, solvent B was 20% AN in
1 mM HCl. Length of gradient from 0 to 100%B was
two column volumes, at flow rate 1 ml/min and UV
detector was set at 268 nm. Enzyme concentration was
100 mg lyophilized enzyme (specific activity of
25 U/mg) in 1 ml reaction mixture. 1U of enzyme
activity was defined as the amount of enzyme that libꢀ
hydroxybenzyl alcohol (A) in transglucosylation reaction,
i.e. “constant of decomposition” complex [E–G + A];
V
maxs
kcat = ꢀꢀꢀꢀꢀꢀꢀꢀ
s
E0
(2)
k3k4(k5k6[GA] + k1k6[M] + k–5k1[M])
=
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ ,
erates 1
nitrophenylꢀ
Determination of kinetic parameters for hydrolysis
ꢀisosalicin. Michaelis constant ( m) for secondary
hydrolysis (Fig. 1), was obtained using 4ꢀnitrophenylꢀ
ꢀDꢀglucopyranoside (0.45 mM) as one substrate and
μ
mol of glucose at 25°C per 1 min from 4ꢀ
(coefA) + (coefGA ⋅ A)[GA] + (coefM)[M]
where kcat represents turnover number for reaction of
α
ꢀDꢀglucopyranoside.
s
α
ꢀisosalicin synthesis, i.e. catalytic rate constant for
conversion of complex [E–G + A] in free enzyme and
ꢀisosalicin (GA). In practical view, kcat is molecular
of
α
K
α
α
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STUDY OF THE KINETIC PARAMETERS
2319
9.10
7.52
A, mAU
200
160
120
80
GA
A
100%
cB
40
0%
0
0
2
4
6
8
10
, ml
V
Fig. 2. One of the HPLC chromatograms for transglucosylation reaction with orthoꢀhydroxybenzyl alcohol (A, retention volume
9.10 ml) as glucose acceptor and ꢀisosalicin (GA, retention volume 7.52 ml) as product of reaction. Yield of reaction was
α
calculated from peak area ratio at 268 nm between GA and A. This chromatogram is obtained when initial concentration of
orthoꢀhydroxybenzyl alcohol was 30 mM and reaction was stopped after 30 min. Conditions for HPLC chromatography are
described in EXPERIMENTAL section.
activity of enzyme and represent number of molecules [A] is concentration of orthoꢀhydroxybenzyl alcohol;
of substrate that one molecule of enzyme transforms in
one second. Knowing total concentration of enzyme
(E0) and Vmax is easy to calculate kcat (Eq. 2). Values for
[GA] is concentration of
tration of maltose.
α
ꢀisosalicin; [M] is concenꢀ
The product formation rate (
d[GA]/dt) for each
specific “coef” and “const” are represented below:
substrate concentration was calculated from HPLC
chromatograms (Fig. 2) Michaelis constant and kcat
are determined from Lineweaver–Burk (LB) plot,
derived from Michaelis–Menten (MM) plot (Fig. 3).
const = k2(k6 + k–5)(k4 + k–3),
(coefA) = k3k4(k–5 + k6),
(coefGA) = k5(k4 + k–3)(k6 + k2),
(coefM) = K1(k6 + k–5)(k4 + k–3),
(coefGA ⋅ A) = k3k5(k4 + k6),
(coefA ⋅ M) = k3K1(k–5 + k6);
Determination of kinetic parameters for hydrolysis
of αꢀisosalicin. Like in case of synthesis, using King
and Altman modified Michaelis–Menten equation
based on Fig. 1, following kinetic parameters are given
for secondary hydrolysis of created product as:
const + (coefA)[A] + (coefM)[M] + (coefA ⋅ M)[A][M]
Km = ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ,
(3)
h
(coefGA) + (coefGA ⋅ A)[A]
experiments measuring the reaction rate,
tion of the substrate starting concentration. In a wellꢀ
known manner, which we used to described synthesis.
V
, as a funcꢀ
where Km represent Michaelis constant for [E + GA]
h
analog that for the synthesis reaction,
K
m and Vmax can be obtained by plotting 1/V in a funcꢀ
V
k5k6(k3k4[A] + k2k4 + k–3k2)
(coefGA) + (coefGA)[A]
maxh
kcat = ꢀꢀꢀꢀꢀꢀꢀꢀꢀ = ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ, (4)
tion of 1/[S] (the LB plot). To determine the reaction
rate by graphical differentiation of the substrate conꢀ
centrationꢀtime curve, at least several experiments
must be performed. This procedure is not only tedious
but also inaccurate, especially if a too high substrate
conversion during the measurement causes a flat curve
instead of a straight line in the substrate concentraꢀ
h
E0
where kcat is turnover number for [E + GA] and also
h
has analog meaning as in case of synthesis.
The specific “Coef” and “const” are the same as in
explanation of synthetic reaction.
Michaelis constant was determined using one of tionꢀtime course [14–17]. The integrated form of
integrated forms of Michaelis–Menten equation [14]. Michaelis–Menten method for enzyme kinetic
The traditional technique for determination of parameter determination is direct and more conveꢀ
enzyme kinetic parameters is to perform numerous nient. This method enables establishment of Km and
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
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ꢀ
C
VELI KOVI C et al.
2320
strate used, and therefore allows the use of concentraꢀ
tion that are far below m. In this way it is possible to
use only one, an arbitrary, concentration of substrate,
thus saving the total consumption of high cost subꢀ
V0
,
µ
M/min
50
40
30
20
10
K
0.07
strate, like
stants.
αꢀisosalicin is, in determination of the conꢀ
0.06
0.05
0.04
0.03
0.02
As the maltase is “the most active” toward 4ꢀnitroꢀ
phenylꢀ ꢀDꢀglucopyranoside [18], so any other subꢀ
strate can be considered as competitive “inhibitor” for
hydrolysis 4ꢀnitrophenylꢀ ꢀDꢀglucopyranoside [19].
Applying Eq. (5) to calculate m and max for 4ꢀnitroꢀ
phenylꢀ ꢀDꢀglucopyranoside, values obtained matched
α
0
10
20
[S], mM
30
40
α
K
V
α
literature values [18] (Fig. 4).
The results obtained for 4ꢀnitrophenylꢀ ꢀDꢀgluꢀ
α
copyranoside in presence of
Fig. 5.
α
ꢀisosalicin are shown in
0
0.1
0.2
0.3
0.4
0.5
1/[S], mMꢁ1
As it was expected, Km for hydrolysis 4ꢀnitropheꢀ
nylꢀ ꢀDꢀglucopyranoside increased (for factor ) and
α
α
Fig. 3. LB and MM plot for synthesis of
[M] = 1.2 M; Km = 6.62 mM, = 0.616 μM/(min mg).
α
ꢀisosalicin;
the Vmax remained the same, suggesting that it is a
competitive inhibition [11]. In the case of the competꢀ
itive inhibition
V
max
s
Kampp
1 + [I]
V
max, by only one experiment, in which the change of
α = ꢀꢀꢀꢀꢀꢀꢀ = ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ,
(6)
substrate concentration in time is monitored:
Km
Ki
where [I] is concentration of inhibitor, i.e. in our case
Km c0 + Km
τ
1
1
⎛
⎞
ꢀꢀ = ꢀꢀꢀꢀꢀꢀꢀ ꢀꢀlnꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ – 1 + ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ ,
(5)
α
ꢀisosalicin, and i is inhibition constant, i.e. “decomꢀ
K
⎝
⎠
U
Vmax
U
1 – U
Vmax
position constant” for enzyme inhibitor complex, or on
Fig. 1, “decomposition constant” for [E + GA] comꢀ
plex. Because “decomposition constant” for [E + GA]
where
τ
is the time point of the reaction,
strate conversion, and c0 is initial substrate concenꢀ
U
is subꢀ
complex actually represents Km for hydrolysis ꢀisosꢀ
α
tration [14].
h
alicin, Km and Ki can be equated. Using Eq. (6) we
Results obtained using this form of integrated
equation do not depend on the concentration of subꢀ
h
calculated that Km is 0.61 mM.
h
τ/U, s
τ/U, s
1400
1200
1000
800
1000
900
800
700
600
600
0
1
2
3
4
0.5
1.0
1.5
(1/
2.0
)ln(1/(1
2.5
))
(1/U)ln(1/(1 − U)) − 1
U
−
U
−
1
Fig. 5. Plot of integral form for Michaelis–Menten equaꢀ
tion for 0.45 mM 4ꢀnitrophenylꢀ ꢀDꢀglucopyranoside as
substrate in the presence of 0.35 mM
α
Fig. 4. Plot of integral form of Michaelis–Menten equaꢀ
tion for 0.45 mM 4ꢀnitrophenylꢀ ꢀDꢀglucopyranoside
as substrate without inhibitor; = 0.27 mM,
25 M/(min mg). Concentration of enzyme used was
75 U/ml.
α
ꢀisosalicin as the
α
app
second substrate (i.e. inhibitor), Kampp = 0.45 mM,
V
=
max
K
V
=
max
m
μ
25 μM(min mg). Concentration of enzyme used was
100 U/ml.
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
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2011

STUDY OF THE KINETIC PARAMETERS
2321
Kinetic constants for transglucosylation reaction catalyzed
by maltase
maltose drops to value comparable to
this point the reaction should be stopped before the
system enters thermodynamic equilibrium.
α
ꢀisosalicin. At
A isosalicin
synthesis
Constant
m, mM
Vmax mol/(min mg)
cat, s–1
hydrolysis
ACKNOWLEGMENTS
K
6.62
0.616
2.5
0.61
8.30
This study was supported by Ministry of Science of
Republic of Serbia, project no. 172049.
,
μ
k
33.67
55.2
K
spec, s–1/mM–1
0.38
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Note:
K
represents real measure of affinity of some enzyme
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cat
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CONCLUSION
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activity of a glucosidase from Saccharomyces cerevisiae
was examined. Also this is the first time ever that by
using kinetic parameters, secondary hydrolysis of
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ꢀglucosidase was described,
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although it is crucial for maximal yield of transglucoꢀ
sylation. Constants calculated in our study are derivaꢀ
tive from our experimental condition, but it is obvious
(Eqs. (1)–(4)) that in other concentrations of malꢀ
tose, or maltose and alcohol in case of hydrolysis,
these constants are different.
12. R. Prodanovicꢂ, N. Milosavicꢂ, D. Sladic, M. Zlatovicꢂ
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otics 33 (3), 293 (1979).
α
16. I. A. Nimmo and G. L. Atkins, Biochem. J. 141, 913
It would be wrong to conclude that when concentraꢀ
tion of the product reaches near 1 mM and concentraꢀ
tion of alcohol is 10 times smaller than product, secꢀ
ondary hydrolysis starts. It should be emphasized that
large amount of maltose is present in the reaction mixꢀ
ture (1.2 M), and as Km for maltose is 80 mM [18]
(1974).
17. G. H. M. Counotte and R. A. Prins, Appl. Environ.
Microbiol. 38 (4), 758 (1979).
18. K. Yamamoto, A. Nakayama, Y. Yamamoto, and
S. Tabata, Eur. J. Biochem. 271, 3414 (2004).
19. E. V. Eneyskaya, A. M. Golubev, A. M. Kachurin,
A. N. Savel’ev, and K. N. Neustroev, Carbohydr. Res.
305, 83 (1998).
enzyme practically “does not feel” the ꢀisosalicin.
α
Secondary hydrolysis starts when concentration of
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
Vol. 85
No. 13
2011