The Reaction Mechanism and Kinetics Data of Racemic Atenolol Kinetic Resolution via Enzymatic Transesterification Process Using Free Pseudomonas fluorescence Lipase

Article abstract of DOI:10.1002/kin.20986

A thorough study on free-enzyme transesterification kinetic resolution of racemic atenolol in a batch system was investigated to gain knowledge for (S)-atenolol kinetics. Analyses of enzyme kinetics using Sigma-Plot 11 Enzyme Kinetics Module on the process are based-on Michaelis-Menten and Lineweaver-Burk plot, which give first-order reaction and ordered-sequential Bi-Bi mechanism, where V_max, K_{M-vinyl acetate}, and K_{M-(S)-atenolol} are 0.80 mM/h, 29.22 mM, and 25.42 mM, respectively. Further analyses on enzyme inhibitions find that both substrates inhibit the process where (S)-atenolol and vinyl acetate develop competitive inhibition and mixed inhibition, respectively. Association of (S)-atenolol with free enzyme to inhibit the enzyme is higher than reaction of active enzyme-substrate complex with vinyl acetate.

Full text of DOI:10.1002/kin.20986

The Reaction Mechanism
and Kinetics Data of
Racemic Atenolol Kinetic
Resolution via Enzymatic
Transesterification Process
Using Free Pseudomonas
fluorescence Lipase
JONI AGUSTIAN,1 AZLINA HARUN KAMARUDDIN2
1Department of Chemical Engineering, Universitas Lampung, Bandar Lampung, 35145, Lampung, Indonesia
2School of Chemical Engineering, Universiti Sains Malaysia, 14300 Nibong Tebal, Seberang Perai Selatan,
Penang, Malaysia
Received 18 August 2015; revised 16 December 2015; accepted 8 February 2016
DOI 10.1002/kin.20986
Published online 15 March 2016 in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: A thorough study on free-enzyme transesterification kinetic resolution of racemic
atenolol in a batch system was investigated to gain knowledge for (S)-atenolol kinetics.
Analyses of enzyme kinetics using Sigma-Plot 11 Enzyme Kinetics Module on the process
are based-on Michaelis–Menten and Lineweaver–Burk plot, which give first-order reaction
and ordered-sequential Bi–Bi mechanism, where Vmax, KM-vinyl acetate, and KM-(S
are
)-atenolol
0.80 mM/h, 29.22 mM, and 25.42 mM, respectively. Further analyses on enzyme inhibitions find
that both substrates inhibit the process where (S)-atenolol and vinyl acetate develop competi-
tive inhibition and mixed inhibition, respectively. Association of (S)-atenolol with free enzyme
to inhibitꢀCthe enzyme is higher than reaction of active enzyme–substrate complex with vinyl
acetate.
2016 Wiley Periodicals, Inc. Int J Chem Kinet 48: 253–265, 2016
INTRODUCTION
Correspondence to: Azlina Harun Kamaruddin; e-mail:
Atenolol chemically known as 2-{4-[2-hydroxy-3-
(propan-2-ylamino)propoxy]phenyl} acetamide is a
chazlina@usm.my.
ꢀC
2016 Wiley Periodicals, Inc.

254
AGUSTIAN AND KAMARUDDIN
β1-selective β-adrenergic receptor blocking drug used
to treat chest pain, hypertension, and heart attack. The
drug is recognized as one of the best-selling drugs in
the world [1–3]. It is mainly sold in the form of racemic
mixture. This β-blocker depends on its β-blocking ac-
tivity on (S)-enantiomer. The eudismic ratio of the
compound is 46 [4]. Because (R)-atenolol has no β-
blocking activity and brings side effects [5,6], switch-
ing from the racemic atenolol to (S)-atenolol produces
lesser side effects [7].
is made available. However, no report on free enzyme-
based racemic atenolol kinetic resolution was found.
Therefore, we studied the kinetic resolution of racemic
atenolol via enzymatic transesterification using free
Pseudomonas fluorescence lipase (Amano) and vinyl
acetate in tetrahydrofurane to gain knowledge for the
(S)-atenolol preparation. Herewith, the kinetics study
of the resolution process is reported since determina-
tion of the kinetics data is important for better under-
standing and scaleup of the resolution process [22].
Two ways are developed to make the (S)-atenolol:
asymmetric synthesis and resolution of the avail-
able racemic atenolol. The asymmetric synthesis via
the chemo-catalysis process required chiral chemi-
cal catalysts such as bimetallic chiral [Co(salen)]-
type complexes to be used to convert racemic or
achiral substrates hydrolytically or directly [8–11].
The compound was also synthesized asymmetrically
via the chemo-enzymatic catalysis process of 4-
hydroxyphenylacetamide and chiral epichlorohydrin
using NaOH and immobilized Candida antarctica li-
pase A (CAL-A CLEA) as the catalysts, which was
claimed as the greener process over other methods [12].
It was made by reacting achiral feeds with the avail-
ability of chiral addenda as well [13–15]. Although
the asymmetric syntheses provide outstanding choices,
they frequently use low reaction temperature and long
reaction steps. Besides, the chiral catalysts are still ex-
pensive.
EXPERIMENTAL
Materials
The (R,S)-atenolol (99% USP) purchased from Nanjing
Chemlin Chemical Industry (Nanjing, People’s Repub-
lic of China) was used. The calibration curves were pre-
pared from the (S)-atenolol (97%; Tocris Bioscience,
Bristol, UK) and (R)-atenolol (99%; Sigma Aldrich,
Selangor, Malaysia). The chemicals were of analytical
grade except for analysis (of HPLC grade) supplied by
EOS Scientific (Selangor, Malaysia), Fisher Scientific
(Selangor, Malaysia), and Merck (Selangor, Malaysia).
Pseudomonas florescence lipase (Amano, product no.
534730, 20 U/mg) was bought from Modern-Lab
Chemicals (Penang, Malaysia). The racemate, single
enantiomers, lipase, and all chemicals were used with-
out pretreatment.
The second way uses the existing racemic atenolol
as the substrate for the (S)-atenolol formation. The
racemate was resolved via microbial (whole-cells) fer-
mentation, which gave high yield and enantiomeric
excess [16], enzymatic kinetic resolution using im-
mobilized lipases [17,18], or liquid chromatography
process using specific chiral selectors [19,20]. The
fermentation process should consider preparation and
recovery of the whole cells when this technology is
applied. The liquid chromatography frequently needs
tedious sample preparations, expensive chiral columns,
and large volumes of additives [21]. The kinetic res-
olution process using immobilized enzymes utilized
diatomite earth and Eupergit–Carbon as the enzyme
support. These immobilized enzymes need a sort of
enzyme recycle system to reuse the enzyme in the
process.
Nowadays, enzymes can be immobilized onto the
membrane surfaces to form enzymatic membrane reac-
tors (EMR) that can operate continuously and be used
repeatedly. They could give a better prospect on the
racemic atenolol resolution. Proper enzyme(s) must
first be chosen as the EMR require free enzyme(s) to
be attached onto the surfaces. Hence, screening of free
enzymes must be conducted before the EMR process
Procedure and Analysis
The racemic atenolol and vinyl acetate concentration
were set in the range of 3.75–30.04 and 43.4–86.8
mM, respectively. Both concentrations were acquired
from the previous experiments (the data are not shown).
The concentration of the racemate was kept constant
when vinyl acetate quantities were varied and vice
versa. Fifty milliliters of 18.80 mM racemic atenolol
dissolved in tetrahydrofurane was prepared in some
100 mL flasks. Then vinyl acetate at different quanti-
ties was mixed into each flask. After the flasks were
shaken for 30 min, every flask was added with enzyme.
All experiments used operating conditions obtained
from the optimization step (45°C, 190 rpm, 2000 U
Pseudomonas flurescence lipase (PFL)). The mixtures
were incubated in orbital shakers (Max Q4000 Barn-
stead Lab-Lin). Samples were taken on certain time
interval and were analyzed chromatographically. The
data were processed using Sigma-Plot 11 Enzyme Ki-
netics Module.
Atenolol enantiomers were observed using
ChiralcelꢀR
OD column (250 mm × 4.6 mm)
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REACTION MECHANISM AND KINETICS DATA OF RACEMIC ATENOLOL KINETIC RESOLUTION
255
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0
2
4
6
8
10
12
Reaction Time (hour)
3.76 mM (S)
3.76 mM (R)
22.53 mM (S)
11.26 mM (S)
22.53 mM (R)
11.26 mM (R)
30.04 mM (S)
18.77 mM (S)
30.04 mM (R)
18,77 mM (R)
Figure 1 Conversion of both enantiomers at various atenolol concentrations (dashes: (R)-atenolol; line in bold: (S)-atenolol).
installed on a Shimadzu LC-20A Prominence Ul-
tra Fast Liquid Chromatograph (UFLC). The mo-
bile phase consisted of hexane–ethanol–diethyl amine
(75%:25%:0.1% v/v) and was flowed at 0.5 mL/min.
A UV/Vis detector was set at the wavelength of
276 nm. The UFLC was operated at the normal phase
at 35°C. 2 L samples were injected directly without
pretreatment. Under these conditions, the (R)-atenolol
was detected at minute 14.1, whereas the (S)-atenolol
was observed at minute 16.7.
tion. Under the racemic atenolol concentrations of
11.26–30.04 mM, less than 60% of the (S)-atenolol
was changed, but the conversions increased steadily at
the concentration range. In contrast, only as high as
18.50% of the (R)-atenolol reacted with vinyl acetate,
which was obtained at the lowest racemic atenolol con-
centration. Reduction of conversion of the (R)-atenolol
was observed when the racemic atenolol concentration
was increased.
Several factors could cause the decrease of conver-
sion when the racemic atenolol concentration was in-
creased. At low substrate concentrations, as the enzyme
activity was constant throughout the trials, many en-
zyme active sites were available for the substrates. But,
at high racemic atenolol concentrations, many acetyl–
enzyme complexes were formed and many vinyl ac-
etate molecules still existed in the solution as constant
enzyme quantity was used. Hence, the reaction envi-
ronment was enriched with the (R)- and (S)-atenolol
and vinyl acetate as constant reaction volume was
applied. Romero et al. [23] described that properties
of medium (e.g., polarity) were modified when al-
cohol and acetic anhydride concentrations were in-
creased leading to the reaction that may be shifted
to the polarest substrate(s), and the free amount of
substrates in the medium caused enzyme deactivation.
Another possible factor is that the amount of enzyme
became the limiting substance at higher substrate con-
centration [24]. The same results were observed during
RESULTS AND DISCUSSION
Conversion of the (S)- and (R)-atenolol during the ini-
tial reaction rate observation at various substrate con-
centrations is shown in Figs. 1 and 2. The enzyme pre-
ferred the (S)-enantiomer. Although both enantiomers
were changed during the reaction, lower conversions
of the (R)-atenolol were obtained. In general, 2.69–
87.14% of the (S)-atenolol and 0–18.47% of the (R)-
atenolol were converted. The conversions of both enan-
tiomers were high at low substrate concentrations. The
highest conversion of the (S)-atenolol was obtained at
the lowest racemic atenolol concentration (3.76 mM).
At this condition, around 87% of the enantiomer was
converted. However, the conversion of this enantiomer
decreased sharply at the highest substrate concentra-
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256
AGUSTIAN AND KAMARUDDIN
50%
45%
40%
35%
30%
25%
20%
15%
10%
5%
0%
0
2
4
6
8
10
12
Reaction Time (hour)
(1:2.4) (S)
(1:2.4) (R)
(1:3.0) (S)
(1:3.0) (R)
(1:3.6) (S)
(1:3.6) (R)
(1:4.2) (S)
(1:4.2) (R)
(1:4.8) (S)
(1:4.8) (R)
Figure 2 Conversions of enantiomers at different vinyl acetate supply (dashes: (R)-atenolol; line in bold: (S)-atenolol).
racemic propranolol resolution using Candida antarc-
tica lipase B (CALB) [18]; however, resolution of the
racemic atenolol using CALB showed increases of con-
version when the racemate concentration was changed
from 3.30 to 16.60 mM [25].
Comparison of the reaction rate of both atenolol
enantiomers is shown in Fig. 3. The transesterifica-
tion reaction rate for the (S)-atenolol is higher than the
(R)-atenolol. The reaction rate of the (S)-atenolol was
4.73–126.32 times higher than that of the (R)-atenolol.
The enantiomeric ratio (E value) of the enzyme in the
kinetic resolution can be calculated from the ratio of
the two reaction rates (the transesterification reaction
rate of the (S)-atenolol over the (R)-atenolol), which
in this case the E values were about 5–62. Since the
(R)-atenolol was found to be less reactive in the ki-
netic resolution, assumption of one substrate and one
product was applied in the enzymatic reaction.
Since the reaction involves two substrates, the com-
pounds ratio in the reaction must be optimized to
give high conversion and to minimize the reactants
cost [23]. When the enzymatic reaction was conducted
under various vinyl acetate concentrations (the sub-
strates’ ratios were set from 1:2.4 to 1:4.8 as described
in Fig. 2), conversions of the (S)-atenolol increased
steadily, which were higher than the (R)-atenolol that
showed little increases. High concentration of vinyl ac-
etate tended to result in high conversion of both enan-
tiomers. The highest conversion of the (S)-atenolol was
found at the highest vinyl acetate concentration that
was the same as the (R)-atenolol. The conversion of
11% was obtained on the (R)-atenolol at the highest
vinyl acetate concentration. The observed ratio fol-
lowed the previous results such as Damle et al. [17]
used the molar ratio of 1–1.2 to change the (R)-atenolol
to ester using vinyl acetate or succinic anhydride
and lipase PS-D to give 40–42% overall yield. Later,
Barbosa et al. [25] used a ratio of vinyl acetate of 2–10
to convert the (R)-atenolol, which obtained the con-
versions of 55–75% where the substrate molar ratio
of 2 produced the best result. Some acetylation-based
enzymatic processes used 1–5 molar ratios [26–30].
Kinetics without Accounting Inhibition
Kinetics of the racemic atenolol transesterification was
first developed without inclusion of the inhibition pro-
cess to justify that the enzymatic reaction was operated
at minimum inhibition effect(s). The kinetics was esti-
mated by using Michaelis–Menten and Lineweaver–
Burk plots on the enzymatic reaction mechanisms.
Theoretically, in the lipase-catalyzed transesterifica-
tion, it has been established that the lipase first forms an
acetyl–enzyme complex with the acetyl donor, which
rules out the random mechanism of the enzymatic
reaction [31–33]. Thus, in estimating the kinetic pa-
rameters using the software, vinyl acetate was entered
first. The schematic diagrams of available enzymatic
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REACTION MECHANISM AND KINETICS DATA OF RACEMIC ATENOLOL KINETIC RESOLUTION
257
Figure 3 Rate of reaction of the (S)- and (R)-atenolol transesterification as a function of racemic atenolol concentration after
10 h reaction.
Table I The Predicted Enzymatic Reaction Mechanisms without Inhibition
Order Bi–Bi
Ping–pong Bi–Bi
E: enzyme; VA: vinyl acetate; (S)-AT: (S)-atenolol; (S)-AT-Ac: (S)-atenolol acetate; Ac: acetate; VOH: vinyl alcohol.
(R2 for good correlations are 70–100% [34–36]. None
article on the racemic compound kinetic resolution was
found mentioning the R2 values on a selected mecha-
nism; hence a comparison cannot be made. The Akaike
information criterion correction (AICc) values on both
mechanisms are excellent as low results are obtained.
The values of the AICc for the ordered-sequential Bi–
Bi and ping–pong Bi–Bi mechanism are around –170.
Low AICc values correspond to better fits the model
to the data [37]. Values of sum of squares for both
mechanisms are <8%, indicating smaller random error
component(s) on the models. The probability values (P
values) are good where they almost approach the sig-
nificance of 5% (the suggested P value in Sigma–Plot
calculation [38]). Therefore, these mechanisms’ mod-
els are acceptable.
reaction mechanisms without any inhibition process
are given in Table I. The Michaelis–Menten graphs for
these mechanisms are shown in Fig. 4. The graph indi-
cates that a first-order reaction occurred. The ordered
Bi–Bi mechanism produces a better graphical repre-
sentation of the reaction rates as parallel lines were
formed within the trial concentrations. The ping–pong
Bi–Bi mechanism showed almost the same rate of re-
action at the (S)-atenolol concentration of less than
2 (two) mM.
Results of the calculation for these mechanisms
produce the same coefficient of determination (R2)
(Table II). Although the R2 are not high (the closer the
value of the R2 to 1 (one), the better the model predicts
the dependent variables), the results are considered ac-
ceptable for kinetic resolution of a racemic compound
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AGUSTIAN AND KAMARUDDIN
Figure 4 Michaelis–Menten plots on various vinyl acetate concentrations as a function of (S)-atenolol concentration.
By comparing the Vmax value, the ping–pong Bi–
Menten graphs within the applied concentrations of
both substrates. Hence, the ordered-sequential Bi–Bi
mechanism was acceptable as its Vmax does not differ
highly with the experimental reaction rates. Values of
the Michaelis constants for the ordered-sequential Bi–
Bi mechanism were lower than the Michaelis constants
for the ping–pong Bi-Bi. Low Michaelis constants are
Bi mechanism gave higher result (4492.99 mmol/L/h)
than the ordered-sequential Bi–Bi mechanism (0.80
mmol/L/h). The Vmax for the ping–pong Bi–Bi mech-
anism exceeds the experimental reaction rates. As can
be seen from Fig. 4, the reaction rates of less than
1 (one) mmol/L/h were used to develop the Michaelis–
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REACTION MECHANISM AND KINETICS DATA OF RACEMIC ATENOLOL KINETIC RESOLUTION
259
Table II Comparison of the Available Mechanisms’
Results
dicted that the inhibition caused by the (S)-atenolol is
competitive type, whereas vinyl acetate shows a mixed
inhibition process. The inhibition caused by vinyl ac-
etate, when its concentrations were increased, was low
as the value of the Vmax decreased slightly. But, (S)-
atenolol produced low effect at the concentration of
less than 9.70 mM as small differences were observed
on Michaelis constant values. At higher (S)-atenolol
concentrations, the inhibition effects increased rapidly.
Based on the inhibition process, new reaction mech-
anism and equation for predicting kinetic constants,
which include the inhibition, must be established.
Ordered-
Sequential
Ping–Pong Nonsequential
Item
Value SEE
Value
SEE
Vmax (mM/h)
Ka (mM)
Kb (mM)
AICc
0.80
0.39
4,492.99
1.46 × 107
29.22 46.65 61,892.92 2.01 × 108
25.42 48.12 5.269 × 105 1.71 × 109
–170.97
75.0%
0.073
0.053
0.064
–170.86
74.9%
0.073
0.052
0.064
R2
Sum of squares
Residual SD
P value
Competitive Inhibition by (S)-Atenolol (A)
mM: millimolar; h: hour; AICc: Akaike Information Criterion
correction; R2: coefficient of determination; SEE: standard error of
estimation; SD: standard deviation, Ka: Michaelis constant for vinyl
acetate; Kb: Michaelis constant (S)-atenolol.
Table III shows the new enzymatic ordered-sequential
Bi–Bi mechanism for the competitive inhibition by the
(S)-atenolol. The rate of reaction is defined as
Vmax
v
KaKb
Kb
KaKb
=
+
+ 1 +
expected to give high enzymatic reaction rates since
these constants are a denominator in mathematical
formulas as shown in the previous equations. As the
standard error of estimation for the ping-pong Bi–Bi
mechanism is extremely high; therefore, the ordered-
sequential Bi–Bi mechanism is the most appropriate
kinetic model for the enzymatic transesterification of
the racemic atenolol using vinyl acetate.
Equation (1) describes the final form of the
racemic atenolol transesterification enzyme kinetics
based on the ordered-sequential Bi–Bi mechanism for
a two substrates reaction without the compound(s)
inhibition:
[VA] [(S) − AT] [(S) − AT]
KI [VA]
(2)
The new forms of Eq. (2) under constant (S)-
atenolol concentration and various vinyl acetate con-
centrations and vice versa are shown in Table IV. To
obtain the inhibition constant, these equations are lin-
earized (y = 1/ν, x = 1/[substrate]). Linear regression
of the reaction rates versus (S)-atenolol concentration
(Fig. 6) gives KI in the intercept portion (Table IV). In
the same way, plot of the reaction rates versus vinyl
Vmax [A] [B]
(0.80) ([VA]) ([(S) − AT])
(1)
v =
=
KaKb + Kb [A] + [A] [B]
742.77 + 25.42 ([VA]) + ([VA]) ([(S) − AT])
acetate concentration results the KI in the slope portion.
As KI is positive, the competitive inhibition by (S)-
Enzyme Kinetics Accounting Inhibition
atenolol indeed occurs.
The presence of the compound(s) that inhibits the en-
zymatic transesterification reaction is confirmed by
Lineweaver–Burk and Eadie–Hofstee plot. The inhi-
bitions caused by the substrates were clearly observed.
However, the inhibition developed by product(s) were
ignored as the (S)-atenolol acetate is not available com-
mercially; hence, it cannot be investigated separately
to know its effect(s). Furthermore, inhibition caused
by (R)-atenolol was also unaccounted.
Identification of the inhibition type through the
Lineweaver–Burk plot for the ordered-sequential Bi–
Bi mechanism is shown in Fig. 5. By comparing both
graphs with literature sources [39,40], it could be pre-
Mixed Inhibition by Vinyl Acetate (B)
The second inhibition type observed in the reaction is
mixed inhibition by vinyl acetate. It is characterized
by attachment of an inhibitor on two positions: to free
enzyme (E) on sites other than the active sites to form
the enzyme–inhibitor complex (EI) and to enzyme–
substrate complex (ES) to give enzyme–substrate–
inhibitor complex (ESI) [41]. There are three possibil-
ities for vinyl acetate to attach to the enzyme to inhibit
the reaction as described in the mechanism shown in
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260
AGUSTIAN AND KAMARUDDIN
Figure 5 Lineweaver–Burk plots of the ordered-sequential Bi–Bi mechanism: (A) various (S)-atenolol concentrations and (B)
various vinyl acetate concentration.
Table III. The rate of reaction is given as below.
Fig. 6) produces KI3 in the intercept portion, whereas
other constants described in the slope part cannot
v
1
=
(3)
KaKb
[VA][(S)−AT]
Kb
[(S)−AT]
KaKb
I1[(S)−AT]
Kb[VA]
[VA]
KI3
Vmax
+
+ 1 +
+
+
I2[(S)−AT]
K
K
be solved. Plotting Eq. (3) against the vinyl acetate con-
centration (y = a*x + b/x + c, y = 1/v, x = [vinyl ac-
etate]; Fig. 7) gives a nonlinear equation that is solved
using Polymath to yield KI2 and KI1 (Table IV). KI2
The forms of Eq. (3) at constant (S)-atenolol con-
centration or vinyl acetate concentration are given in
Table IV. Linear regression of the Eq. (3) against (S)-
atenolol concentration ((y = 1/v, x = 1/[(S)-atenolol];
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REACTION MECHANISM AND KINETICS DATA OF RACEMIC ATENOLOL KINETIC RESOLUTION
261
Table III Enzymatic Reaction Mechanisms for the Inhibition Processes
Inhibition
Code
A
Mechanism
Competitive inhibition
by (S)-atenolol
Mixed inhibition by
vinyl acetate
B
C
D
Both substrates
inhibition
E: enzyme, VA: vinyl acetate, (S)-AT: (S)-atenolol, Ka: Michaelis constant for vinyl acetate, Kb: Michaelis constant for (S)-atenolol, KI:
inhibition constant, P: (S)-atenolol acetate, Q: vinyl alcohol, KI1: inhibition constant 01, KI2: inhibition constant 02, KI3: inhibition constant
ꢁ
ꢁ
03, Ka : modified Michaelis constant for vinyl acetate, Kb : modified Michaelis constant for (S)-atenolol. “*”indicates nonenzyme’ active sites
attachment.
and KI3 are positive meaning the inhibition proceeds
within the pathway.
At various (S)-atenolol or vinyl acetate concentra-
tions, Eq. (4) can be simplified into the equations
shown in Table IV. Under various (S)-atenolol con-
centrations, linear regression of Eq. (4) produces K12
in the slope part. Variations of the vinyl acetate con-
centrations give a nonlinear equation y = a*x + b/x
+ c (y = 1/v, x = [vinyl acetate]; Fig. 7) then solved
by Polymath to give KI1 and KI3. Two positive values
are found (KI1 and KI3) confirming inhibitions indeed
occur following the predicted mechanism.
Enzyme Kinetics with Both Substrates
Inhibitions
There are some mechanisms that can be used to de-
velop the simultaneous inhibition by both substrates
(Table III, C and D). The first alternative (C) uses the
previous mixed mechanism, but free enzyme (E) asso-
ciates with (S)-atenolol to form the dead-end enzyme-
(S)-atenolol complex. Normalization of the reaction
rate gives
Another inhibition mechanism developed to predict
both substrates inhibition is illustrated in pathway D
(Table III). The reaction rate is defined as
v
1
=
(4)
KaKb
[VA][(S)−AT]
Kb
[(S)−AT]
KaKb
Kb[VA]
[VA]
KI3
Vmax
+
+ 1 +
+
+
I2[(S)−AT]
K
I1[VA]
K
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REACTION MECHANISM AND KINETICS DATA OF RACEMIC ATENOLOL KINETIC RESOLUTION
263
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
y = 13.8153x + 1.7656
R² = 0.9926
0.00
0.10
0.20
0.30
0.40
1/[(S]-atenolol (1/mM)
Figure 6 Linear regression plot of (S)-atenolol competitive Inhibition.
0.50
0.60
0.70
0.80
Figure 7 Nonlinear regression plot of vinyl acetate mixed inhibition.
The form of the final equation for the racemic
atenolol transesterification enzyme kinetics based on
the sequential ordered Bi–Bi mechanism for a two-
substrate reaction with mixed inhibition by the sub-
strates is as follows:
v
1
=
KaKb
[VA][(S)−AT]
Kb
[(S)−AT]
KaKb
[VA]
KI3
Vmax
+
+ 1 +
+
K
I1[VA]
(5)
v
[A] [B]
=
When (S)-atenolol or vinyl acetate concentration
is varied, Eq. (5) can be derived to find the inhibi-
tion constants as shown in Table IV. At constant (S)-
KaKb[B]
KI1
[A][A][B]
KI3
Vmax
KaKb + Kb [A] + [A] [B] +
+
(6)
(0.80) (VA) ((S) − AT)
=
(7)
742.77([(S)−AT])
[VA][VA][(S)−AT]
742.77 + (25.42) ([VA]) + ([VA]) ([(S) − AT]) +
+
13.35
129.12
atenolol concentration, a nonlinear equation is formed
(y = a*x + b/x + c; y = 1/v, x = [vinyl acetate]; Fig. 7)
then solved with Polymath nonlinear regression to give
KI1 and KI3. As both inhibition constants are positive,
the possible inhibition mechanism is established.
CONCLUSION
Analysis of the racemic atenolol transesterifica-
tion enzyme kinetics using Michaelis–Menten and
International Journal of Chemical Kinetics DOI 10.1002/kin.20986

264
AGUSTIAN AND KAMARUDDIN
Lineweaver–Burk plot resulted first-order reaction and
ordered-sequential Bi-Bi mechanism with R2: ꢀ75%.
Vmax, KM-vinyl acetate, KM-(S)-atenolol are 0.80 mmol/L/h,
29.22 mM, and 25.42 mM. After observing the inhibi-
tion phenomena using the Lineweaver–Burk plot, both
substrates inhibit the enzymatic reaction where the (S)-
atenolol and vinyl acetate develop the competitive inhi-
bition and mixed inhibition process, respectively. The
inhibition created by the (S)-atenolol was low at low
(S)-atenolol concentrations, but the inhibition effects
increased rapidly at high (S)-atenolol concentrations;
however, vinyl acetate shows a low inhibition effect
throughout the trial concentrations. The inhibition con-
stants for the (S)-atenolol is 13.35 mM whereas for the
vinyl acetate is 129.12 mM.
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Financial supports from Universiti Sains Malaysia
(PRGS: 1001/PJKIMIA/8044030), MOSTI (Science Fund:
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