Kinetics and quantitative structure-activity relationships for pseudomonas sp. Lipase-catalyzed hydrolysis of both monoesters and diesters of ethylene glycol

Article abstract of DOI:10.1007/s11746-006-1194-y

The goal of this work is to study kinetics and quantitative structure-activity relationships for steady states of Pseudomonas sp. lipase-catalyzed hydrolysis of both diesters and monoesters of ethylene glycol. Based on the steady-state kinetics of the enzyme-catalyzed hydrolysis of the diesters of ethylene glycol, the diesters and the monoesters react simultaneously as soon as monoester has started to build up in the reaction medium. In other words, the apparent K_m values of the diesters are the K_m values of the diesters (K_mA) plus the K_m values of the monoesters (K_mB), and all V_max values are about the same. Moreover, the pH-stat titration curve of the enzyme-catalyzed hydrolysis of the diesters of ethylene glycol is initially hyperbolic, then there is a sharp falloff in the hydrolysis rate. The abrupt stoppage of the reaction (relaxation stage) may be due to the existence of two phases in the reaction medium, that is, the product (ethylene glycol) and the substrates (the diesters of ethylene glycol) are not miscible. Furthermore, quantitative structure-activity relationships for varied acyl groups of mono- and diesters of ethylene glycol are studied. The fact that both pK_mA and pK _mB values are linearly correlated with the hydrophobicity constant (π) but not with the electronic substituent constants (σ*) indicates that the affinity of these substrates for the enzyme depends only on the hydrophobicitv of substrates. Copyright

Full text of DOI:10.1007/s11746-006-1194-y

Kinetics and Quantitative Structure–Activity Relationships
for Pseudomonas sp. Lipase-Catalyzed Hydrolysis
of Both Monoesters and Diesters of Ethylene Glycol
Shyh-Ying Chioua, Yu-Ru Chengb, Chun-Ping Luc, Yan-Fu Linb, Long-Yau Lina, and Gialih Linb,*
aInstitute of Medicine and Department of Neurosurgery, Chung Shan Medical University, Taichung 402, Taiwan, and
bDepartment of Chemistry and cInstitute of Biochemistry, National Chung-Hsing University, Taichung 402, Taiwan
ABSTRACT: The goal of this work is to study kinetics and quan-
titative structure–activity relationships for steady states of
Pseudomonas sp. lipase-catalyzed hydrolysis of both diesters and
monoesters of ethylene glycol. Based on the steady-state kinetics
of the enzyme-catalyzed hydrolysis of the diesters of ethylene gly-
col, the diesters and the monoesters react simultaneously as soon
as monoester has started to build up in the reaction medium. In
other words, the apparent Km values of the diesters are the Km val-
ues of the diesters (KmA) plus the Km values of the monoesters
(KmB), and all Vmax values are about the same. Moreover, the pH-
stat titration curve of the enzyme-catalyzed hydrolysis of the di-
esters of ethylene glycol is initially hyperbolic, then there is a
sharp falloff in the hydrolysis rate. The abrupt stoppage of the re-
action (relaxation stage) may be due to the existence of two
phases in the reaction medium, that is, the product (ethylene gly-
col) and the substrates (the diesters of ethylene glycol) are not
miscible. Furthermore, quantitative structure–activity relation-
ships for varied acyl groups of mono- and diesters of ethylene gly-
col are studied. The fact that both pKmA and pKmB values are lin-
early correlated with the hydrophobicity constant (π) but not with
the electronic substituent constants (σ*) indicates that the affinity
of these substrates for the enzyme depends only on the hydropho-
bicity of substrates.
been reported (5–10). Lipase usually contains a small α-helix
or loop, referred to as the lid, that covers the active site pocket
(5,7,8,11–14). This conformation is termed the closed confor-
mation. When the lipase is absorbed to an interface, the lid is
displaced so that the active site becomes accessible to substrate
(15). This conformation is termed the open conformation. The
structures of the free and bound lipase are believed to represent
the start and end conformation in the interfacial activation
process (16,17).
In general, lipases catalyze hydrolysis of TAG to 1,2-DAG,
2,3-DAG, and meso-1,3-DAG, and then also catalyze hydroly-
sis of these three DAG to 1-MAG, meso-2-MAG, and 3-MAG
and then to glycerol. Therefore, the kinetics for lipase-cat-
alyzed hydrolysis of TAG are very complicated owing to the
many intermediates and to different stereoselectivities for some
intermediates. To make the situation simple, we synthesized
both diesters and monoesters of ethylene glycol (1–6 and 7–12,
respectively) as substrates of Pseudomonas sp. lipase (PSL)
and characterized their kinetics.
The first reaction in the PSL-catalyzed hydrolysis of diesters
1–6 is formation of monoesters 7–12 (Fig. 1). Then the enzyme
catalyzes a subsequent reaction in situ to hydrolyze monoesters
7–12 to ethylene glycerol (EG). Thus, the subsequent hydroly-
sis of diesters 1–6 in situ will be the same as the hydrolysis of
monoesters 7–12 to EG.
Figure 1 represents a Ping Pong Bi Bi mechanism. In the ab-
sence of products P and Q, the forward velocity is given by
Equation 1 (18):
Paper no. J11148 in JAOCS 83, 201–207 (March 2006).
KEY WORDS: Enzyme kinetics, esters of ethylene glycol,
Pseudomonas sp. lipase quantitative structure–activity relation-
ship.
The commercial potential of organic syntheses catalyzed by li-
pases (EC 3.1.1.3) underscores the need for a comprehensive
understanding of lipase structure and function and has provided
the impetus for many recent investigations (1,2). Recently,
there has been increased interest in lipases owing to the use of
Orlistat (Xenical®) (3). Orlistat, whose original mechanism of
action consists of the selective inhibition of gastrointestinal li-
pases, has been commercialized for the treatment of obesity.
Lipases are lipolytic enzymes that hydrolyze ester bonds of
TAG (4). However, their substrate specificity is not limited to
TAG. The X-ray structures of many lipases such as
Pseudomonas cepacia lipase and Candida rugosa lipase have
v = Vmax [A] [B]/(KmB [A] + KmA [B] + [A] [B])
[1]
For the hydrolysis of diesters 1–6, the diesters (A) and the mo-
noesters (B) may react simultaneously as soon as some mono-
ester has started to build up in the reaction medium. Then [A]
= [B] and Equation 1 becomes Equation 2.
v = Vmax [A]/((KmA + KmB ) + [A])
[2]
In other words, the apparent Km values of the diesters are the
Km values of the diesters (KmA) plus the Km values of the mo-
noesters (KmB), and the Vmax values are constant.
Furthermore, quantitative structure–activity relationships
(19) for varied acyl substituents of diesters 1–6 and monoesters
*To whom correspondence should be addressed.
E-mail: gilin@dragon.nchu.edu.tw
Copyright © 2006 by AOCS Press
201
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202
S.-Y. CHIOU ET AL.
FIG. 1. Kinetic scheme for the Pseudomas sp. lipase (PSL)-catalyzed hydrolysis of diesters 1–6 to
monoesters 7–12, then the PSL-catalyzed hydrolysis of monesters 7–12 to ethylene glycol. A, the
diesters of ethylene glycol (1–6); B, the monoesters of ethylene glycol (7–12); E, enzyme; EA, en-
zyme-1–6 tetrahedral complex; EB, enzyme-7–12 tetrahedral complex; KmA, Michaelis constant
of 1–6; KmB, Michaelis constant of 7–12; kcatA, turnover number of 1–6; kcatB, turnover number
of 7–12; P, the first product (FA); Q, the second product (ethylene glycol).
1
7–12 may provide us much information to characterize these Compounds 1–12 were characterized by H and 13C NMR
reaction mechanisms.
spectra, mass spectra, and elemental analysis.
Instrumental methods. All kinetic data were obtained from
a pH-stat titration apparatus that was composed of a pH-stat
controller (PHM 290; Radiometer) and an autoburette (AUB
EXPERIMENTAL PROCEDURES
Materials. PSL was obtained from Sigma (St. Louis, MO); 901; Radiometer) with a circulating water bath jacket. 1H and
other chemicals were obtained from Aldrich (Milwaukee, WI). 13C NMR spectra were recorded at 400.45 and 100.70 MHz,
Silica gel used in LC (LiChroprep Silica 60, 200–400 mesh) respectively (Varian Gemini 400 spectrometer). The 1H and
and TLC plates (Kieselgel 60 F254) were obtained from Merck 13C NMR chemical shifts were referred to internal tetramethyl-
(Darmstadt, Germany). A hexane/ethyl acetate solvent gradi- silane. Mass spectra were recorded at 70 eV on a Joel JMS-
ent was used in LC. Other chemicals were of the highest qual- SX/SX-102A mass spectrometer. Elemental analysis was per-
ity available commercially.
formed on a Heraeus instrument.
Synthesis of substrates. The diesters of ethylene glycol (1–6)
Data reduction. Origin (version 6.0; OriginLab, Northamp-
and the corresponding monoesters of ethylene glycol (7–12) ton, MA) was used for both linear and nonlinear least-squares
were synthesized in one-step reactions. The reaction of ethyl- curve fittings.
ene glycol with 2 equiv of acyl chloride in the presence of 2
Enzyme kinetics. All kinetic data were obtained by using a
equiv of sodium hydride (NaH) in THF at 25°C for 24 h yields pH-stat titration apparatus that was interfaced to a personal
diesters 1–6 and the corresponding monoesters 7–12. Diesters computer. Temperature was maintained at 25.0°C by a circu-
1–6 (45–50% yield) and the corresponding monoesters 7–12 lating water bath. All reactions were performed in a NaCl (0.1
(45–50% yield) were then separated and purified by LC. A typ- M) solution with varying concentration of the substrates 1–12.
ical reaction for synthesis of 1,2-dibenzoxy-ethane (4) and 2- Reactions were initiated by injecting PSL into a stock solution
benzoxy-ethanol (10) may be described as follows. Reaction of of substrate. The reactions were titrated from the autoburette,
ethylene glycol with 2 equiv of benzoyl chloride in the pres- which contained 0.001 M NaOH, and the pH-stat controller
ence of 2 equiv of NaH in THF at 25°C for 24 h yields 4 and was set at pH = 8.0. The steady-state initial velocities (v) for
10. Compounds 4 (47% yield) and 10 (45% yield) were sepa- PSL-catalyzed hydrolysis of 1–12 were determined as the ini-
rated and purified by LC.
tial slopes from the initial hyperbolic curves of the time courses
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LIPASE CATALYZES HYDROLYSIS OF THE ESTERS OF ETHYLENE GLYCOL
203
of diesters 1–6 ([A]) plot against Equation 2 (18). Duplicate
sets of data were collected for each substrate concentration.
Relaxation rate (krelax). The relaxation rate (krelax.) for the
PSL-catalyzed hydrolysis of diesters 7–12 was determined as
1/trelax, where trelax is the lifetime of the relaxation stage (Fig.
2).
RESULTS AND DISCUSSION
The diesters of ethylene glycol (1–6) and the monoesters of eth-
ylene glycol (7–12) were synthesized as simple analogs of
TAG, DAG, and MAG.
The v values for the PSL-catalyzed hydrolysis of diesters
1–6 and monoesters 7–12 were obtained from the initial slopes
of the pH-stat titration time courses. The KmB and VmaxB val-
ues for the PSL-catalyzed hydrolysis of monoesters 7–12 were
obtained from the nonlinear least-squares curve fittings of v vs.
substrate concentration plots against the Michaelis–Menten
equation (Table 1). The KmA + KmB and VmaxA values (Table 2)
for the PSL-catalyzed hydrolysis of diesters 1–6 were obtained
from the nonlinear least-squares curve fittings of v vs. substrate
FIG. 2. Time course of pH-stat titration of PSL-catalyzed hydrolysis of 4.
The overall time course consisted of (i) the initial hyperbolic curve and
(ii) the relaxation stage. The reactions were titrated with 0.001 M
NaOH, and the pH-stat controller was set at pH = 8.0. [4] = 0.5 mM. v
= 1.2 µmol/s. The lifetime of the relaxation stage is 50 s. For abbrevia-
tion see Figure 1.
(Fig. 2). The KmB and VmaxB values (Table 1) for PSL-cat- concentration plots against Equation 2 (18). Therefore, KmA
alyzed hydrolysis of monoesters 7–12 were obtained from the was calculated by subtracting KmB from KmA + KmB
nonlinear least-squares curve fitting of v vs. the concentration The pKmA, pKmB, log (VmaxA/KmA), and log (VmaxB/KmB
.
)
of monoesters 7–12 ([B]) plot against the Michaelis–Menten values were linearly correlated with the hydrophobicity con-
equation. The KmA + KmB and VmaxA values (Table 2) for PSL- stant (π) (19) (Fig. 3, Table 3); however, these values were not
catalyzed hydrolysis of diesters 1–6 were obtained from the linearly correlated with the electronic substituent constants σ*
nonlinear least-squares curve fitting of v vs. the concentration (Fig. 4).
TABLE 1
Kinetic Data for Pre-Steady and Steady States of PSL-Catalyzed Hydrolysis
of the Monoesters of Ethylene Glycola (7–12)
Substrate
R
KmB (mM)
VmaxB (nmol/s)
VmaxB/KmB (10-3 mol s-1 M-1)
7
8
9
10
11
12
t-Bu
n-C3H7
CH3
Ph
n-C5H11
n-C7H15
0.15 0.01
1.5 0.2
48.1 0.2
0.32 0.02
0.033 0.004
0.0047 0.0005
0.19 0.05
0.32 0.02
0.48 0.05
49
47
1
1
10
1
0.26 0.07
0.15 0.1
0.10 0.01
48.3 0.7
48.2 0.8
47.5 0.1
aPSL, Pseudomonas sp. lipase.
TABLE 2
Substituent Constants and Steady-State Kinetic Data of PSL-Catalyzed Hydrolysis of the Diesters
of Ethylene Glycol (1–6)
VmaxA
VmaxA/KmA
(10-3 mol s-1 M-1)
Substrate
R
σ* a
πa
KmA (mM)
(nmol/s)
1
2
3
4
5
6
t-Bu
n-C3H7
CH3
Ph
n-C5H11
n-C7H15
−0.3
−0.1
0
0.6
−0.13
−0.13
1.98
1.5
0.5
2.13
2.5
3.5
1.7 0.3
0.30 0.05
3.0 0.3
0.10 0.03
0.19 0.04
0.13 0.04
40.6 0.3
38.9 0.6
0.024 0.004
0.13 0.02
0.015 0.002
0.49 0.01
0.24 0.05
0.4 0.1
45
49
45
47
3
1
1
1
aTaken from References 19–22. σ*, electronic substituent constants; π, hydrophobicity constants; for other abbreviation
see Table 1.
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204
S.-Y. CHIOU ET AL.
FIG. 3. Plots of (A) pKmA, (B) log (VmaxA/KmA), (C) pKmB, and (D) log (VmaxB/KmB) values for PSL-catalyzed hydrolysis of diesters 1–6 and monoesters
7–12 against π, the hydrophobicity constant. Correlation results are summarized in Table 3. For abbreviation see Figure 1.
The pH-stat titration curve of the enzyme-catalyzed hydroly-
Based on the steady-state kinetics of the enzyme-catalyzed
sis of diesters 1–6 consisted of a hyperbolic curve, then a sharp hydrolysis of diesters 1–6 (Table 2), the diesters and the mo-
falloff in the hydrolysis rate (Fig. 2). The abrupt stoppage of the noesters react simultaneously as soon as some monoester has
reaction was a relaxation stage with the rate constant of krelax
.
been built up in the reaction medium. In other words, the appar-
The log krelax values were linearly correlated with π (Fig. 5).
ent Km values of the diesters are the Km values of the diesters
TABLE 3
Results of the Linear Correlations of pKmA, log (VmaxA/KmA), pKmB, log (VmaxB/KmB), and log krelax with π
pKmA
log (VmaxA/KmA
)
pKmB
log (VmaxB/KmB
)
log krelax
ha
2.3 0.3
0.5 0.2
0.813
−4.9 0.5
0.5 0.2
0.740
1.9 0.4
0.7 0.2
0.904
−5.4 0.4
0.7 0.2
0.904
−1.43 0.02
−0.124 0.008
0.992
yb
Rc
SEM
0.187
0.196
0.152
0.152
0.007
aIntercept for the pKmA, log (VmaxA/KmA), pKmB, log (VmaxB/KmB), or log krelax values vs. π plot (Figs. 2, 4).
bSlopes for the pKmA, log (VmaxA/KmA), pKmB, log (VmaxB/KmB), or log krelax values vs. π plot (Figs. 2, 4).
cCorrelation coefficient. For abbreviations see Table 2.
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LIPASE CATALYZES HYDROLYSIS OF THE ESTERS OF ETHYLENE GLYCOL
205
FIG. 4. Plots of (A) pKmA, (B) log (VmaxA/KmA), (C) pKmB, and (D) log (VmaxB/KmB) values for PSL-catalyzed hydrolysis of diesters 1–6 and monoesters
7–12 against electronic substituent constants σ*. For abbreviation see Figure 1.
(KmA) plus the Km values of the monoesters (KmB). The fact that
the VmaxA and VmaxB values (Tables 1 and 2) are about the same
indicates that the PSL-catalyzed hydrolysis of diesters 1–6 pro-
ceeds with the Ping Pong Bi Bi mechanism (Fig. 1) (18). For
the PSL-catalyzed hydrolysis of diesters 1–6, the product of the
first reaction, monoesters 7–12, are instantly excluded from the
active site of the enzyme then quickly diffuse into the active site
(Fig. 6).
The fact that the pKmA, pKmB, log (VmaxA/KmA), and log
(VmaxB/KmB) values were linearly correlated with π (Fig. 3,
Table 3) but not with σ* (Fig. 4) indicates that the PSL-cat-
alyzed hydrolysis of diesters 1–6 and monoesters 7–12 only de-
pends on the hydrophobicity of the substrates. The ψ value (i.e.,
the slope of the log k vs. p plot) of 0.5 or 0.7 for the pKmA- or
pKmB-π-correlation (Table 3) implies that the enzyme–substrate
FIG. 5. Plot of the log krelax. values for PSL-catalyzed hydrolysis of di-
esters 1–6 against π. For abbreviation see Figure 1.
tetrahedral intermediates are more stabilized by a hydrophobic
acyl moiety than a hydrophilic acyl moiety (20–23). This is
probably due to strong van der Waals’ or hydrophobic interac-
tions between the acyl moiety of the substrate and the acyl chain
binding site (6) of the enzyme (Fig. 6). The fact that the ψ val-
ues for the log (VmaxA/KmA)–π-correlation and the pKmA–π-cor-
relation (Table 3) are about the same suggests that the ψ value
JAOCS, Vol. 83, no. 3 (2006)

206
S.-Y. CHIOU ET AL.
FIG. 6. Proposed mechanism for PSL-catalyzed hydrolysis of diesters 1–6 to ethylene glycerol.
The first step (KmA) is formation of the enzyme–diester tetrahedral complex. The second step
(kcatA) involves formation of monoester 7–10 as the first product of the reaction and then for-
mation of the second product, FA. The third step is diffusion of the monoesters 7–10 into the
active site of the enzyme. The fourth step (KmB) involves the formation of the enzyme–mono-
ester tetrahedral complex. The fifth step (kcaB) involves the formation of ethylene glycol. ψ,
slope for log k vs. π plot; ACS, acyl chain binding site; for other abbreviation see Figure 1.
of the reaction (relaxation stage) may be due to a change in the
physical state of the reaction medium. In other words, the re-
laxation stage may be due to the existence of two phases in the
reaction medium, that is, the product (EG) and the substrates
(diesters 1–6) are not miscible (Fig. 7). Furthermore, the log
krelax values are correlated with π (Fig. 5, Table 3). The ψ value
of −0.124 for the above correlation indicates that more hy-
drophobic diesters such as 5 and 6 have longer relaxation times
than less hydrophobic diesters such as 2 and 3.
for the log kcatA–π-correlation is about zero (Fig. 6). Since the
kcatA step consists of two steps, formation of the acyl enzyme
from the tetrahedral intermediate and hydrolysis of the acyl en-
zyme to products, the ψ values for above two steps seem to can-
cel each other. For similar reasons, the ψ value for the log
kcatB–π-correlation is about zero (Fig. 6).
Moreover, the pH-stat titration curve of PSL-catalyzed hy-
drolysis of diesters 1–6 consists of a hyperbolic curve, then a
sharp falloff in the hydrolysis rate (Fig. 2). The abrupt stoppage
JAOCS, Vol. 83, no. 3 (2006)

LIPASE CATALYZES HYDROLYSIS OF THE ESTERS OF ETHYLENE GLYCOL
207
FIG. 7. Proposed mechanism for the relaxation process in the PSL-catalyzed hydrolysis of di-
esters 1–6. The first step is hydrolysis of diesters 1–6 (substrate A). The second step involves
diffusion of ethylene glycerol (EG) out of the active site to form a biphasic medium. In this
medium, substrate A cannot enter the active site of the enzyme owing to the hindrance at the
mouth of the enzyme by the aggregation of EG. The third step (krelax.) is the relaxation process,
which breaks down the biphasic medium. Therefore, substrate A enters the active site of the
enzyme following this relaxation or emulsification.
Jensen, L. Norskov, L. Thim, and U. Menge, A Serine Protease
Triad Forms the Catalytic Center of a Triacylglycerol Lipase,
Nature 343:767–770 (1990).
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JAOCS, Vol. 83, no. 3 (2006)