Substrate conformations set the rate of enzymatic acrylation by lipases

Article abstract of DOI:10.1002/cbic.200900758

Acrylates represent a class of α,β-unsaturated compounds of high industrial importance. We investigated the influence of substrate conformations on the experimentally determined reaction rates of the enzyme-catalysed transacylation of methyl acrylate and derivatives by ab initio DFT B3LYP calculations and molecular dynamics simulations. The results supported a least-motion mechanism upon the sp² to sp³ substrate transition to reach the transition state in the enzyme active site. This was in accordance with our hypothesis that acrylates form productive transition states from their low-energy s-sis/s-trans conformations. Apparent k_cat values were measured for Candida antarctica lipase B (CALB), Humicola insolens cutlnase and Rhizomucor miehei lipase and were compared to results from computer simulations. More potent enzymes for acryltransfer, such as the CALB mutant V190A and acrylates with higher turnover numbers, showed elevated populations of productive transition states.

Full text of DOI:10.1002/cbic.200900758

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DOI: 10.1002/cbic.200900758
Substrate Conformations Set the Rate of Enzymatic
Acrylation by Lipases
Per-Olof Syrꢀn and Karl Hult*[a]
Acrylates represent a class of a,b-unsaturated compounds of
high industrial importance. We investigated the influence of
substrate conformations on the experimentally determined re-
action rates of the enzyme-catalysed transacylation of methyl
acrylate and derivatives by ab initio DFT B3LYP calculations
and molecular dynamics simulations. The results supported a
least-motion mechanism upon the sp2 to sp3 substrate transi-
tion to reach the transition state in the enzyme active site. This
was in accordance with our hypothesis that acrylates form pro-
ductive transition states from their low-energy s-sis/s-trans con-
formations. Apparent kcat values were measured for Candida
antarctica lipase B (CALB), Humicola insolens cutinase and Rhi-
zomucor miehei lipase and were compared to results from
computer simulations. More potent enzymes for acryltransfer,
such as the CALB mutant V190A and acrylates with higher
turnover numbers, showed elevated populations of productive
transition states.
Introduction
Acrylates are a,b-unsaturated esters that are used as mono-
meric starting materials for a wide range of commercial appli-
cations such as coatings, plastics and paints. The double bond
present in this class of molecules is useful for cross-linking pur-
poses by radical polymerisation. Esters of acrylic acid or metha-
crylic acid are traditionally made chemically under harsh reac-
tion conditions with sulfuric acid as catalyst.[1] It is necessary to
add polymerisation inhibitors for processes run at elevated
temperatures.
Scheme 1. The two possible acrylate conformers.
In this study we have investigated the distribution of differ-
ent substrate conformers of methyl acrylate and derivatives by
using ab initio calculations. The influence of substrate confor-
mational distribution on the experimentally determined reac-
tion rate of enzyme-catalysed transacylation was investigated
by molecular dynamics simulations. Thus both the relative
abundance of free substrate conformers and how well these
conformers fit in the active site were considered. CALB wt (EC
3.1.1.3) and mutant V190A, Rhizomucor miehei lipase (RML, EC
3.1.1.3) and cutinase from Humicola insolens (HiC, EC 3.1.1.74)
were analysed. The aim of this work was to get a deeper
understanding of enzyme-catalysed acryltransfer. The gained
knowledge can be used for in silico protein design of lipase
variants with increased activity towards acrylate substrates as a
complement to traditional laboratory work (mutant library con-
struction and screening). The results were in agreement with
our hypothesis that acrylates mainly react in their low-energy,
ground state, s-cis/s-trans conformations.
Biocatalysis is a “green”, sustainable alternative to traditional
chemistry. Enzymes can be used under mild reaction condi-
tions and they show high activity and regio- and stereo selec-
tivity. Lipases are of industrial interest due to their high stabili-
ty in organic media, substrate tolerance and stereoselectivity.[2]
Regioselective transacylation of ethyl acrylate with sterically
hindered diols to obtain the monoester by using lipase from
Chromobacterium viscosum has been described in the litera-
ture.[3] Lipase B from Candida antarctica (CALB, also known as
Pseudozyma antarctica lipase B) showed the best catalytic per-
formance in the transacylation of methyl acrylate with unde-
can-1-ol out of 19 enzymes tested.[4] Monoacrylation of polyols
with CALB has been described in a patent from BASF.[5] The
influence of solvent choice and water activity on the reaction
rate of lipase-catalysed transacylation of acrylic acid and ethyl
acrylate has been reported.[6] One problem with chemical and
enzymatic transacylations of acrylates is the low reaction rate.
To our knowledge, no attempts have been made to explain
this fact.
[a] P.-O. Syrꢀn, Prof. K. Hult
Acrylates have been shown to exist in two conformations, s-
cis and s-trans, Scheme 1, with a high rotational barrier to iso-
merisation.[7] This can be explained by the stabilising inter-
action between the p orbitals of the alkene group and the car-
bonyl double bond.
Department of Biochemistry, School of Biotechnology
Royal Institute of Technology, AlbaNova University Center
10691 Stockholm (Sweden)
Fax: (+46)8-5537-8468
E-mail: kalle@biotech.kth.se
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.200900758.
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Rate of Enzymatic Acrylation
Results and Discussion
Table 2. Population analysis of substrates by using DFT at the B3LYP
6ꢀ311+G(2d,2p) level of theory.
The view on how enzymes can achieve their remarkable rate
acceleration compared to the corresponding reaction in solu-
tion has changed a lot since the lock-and-key model was intro-
duced by Emil Fisher.[8] Clearly, enzymes are not rigid catalysts,
and the important factors for catalysis include proximity ef-
fects, electrostatic stabilisation of the transition state (TS), sym-
metrical hydrogen bonds and tunnelling.[9] For acrylates there
is a stabilising p system in the free substrate that can affect
catalysis. Our hypothesis that the reaction rate for enzyme-cat-
alysed transacylation of acrylates is determined by the ability
of ground state s-cis/s-trans conformations of the free acrylate
substrates to form transition states was evaluated by kinetic
experiments and computer modelling.
[a]
Erel,PCM [kcalmolꢀ1
s-cis
]
Relative abundance[b]
s-trans
s-cis
s-trans
1
2
3
0.0
0.6
0.0
0.6
0.0
0.2
0.73
0.27
0.58
0.27
0.73
0.42
[a] The relative energies with solvation were taken into account by using
the CPCM polarisable continuum model with diethyl ether (e=4.335).
[b] Relative abundances are given at 302 K in diethyl ether.
The experimentally determined apparent kcat values for
transacylation with the saturated ester methyl propionate and
the a,b-unsaturated compounds methyl acrylate, methyl meth-
acrylate and methyl a-chloroacrylate in diisopropyl ether at a
concentration of propan-1-ol of 25 mm are given in Table 1.
A DFT B3LYP 6ꢀ31G(d,p) single-point calculation on methyl ac-
rylate with perpendicular p orbitals resulted in an energy that
was 7 kcalmolꢀ1 higher than that of the low-energy s-cis con-
formation. The single-point energy was thus in good agree-
ment with the calculated barrier at the higher level of theory.
The corresponding single-point energies for methyl methacry-
late and methyl a-chloroacrylate with perpendicular p orbitals
were found to be 6 kcalmolꢀ1 higher than the energies for
their respective low-energy conformation (see Table S5 in the
Supporting Information). The calculated barriers for isomerisa-
tion were thus found to be much smaller than the barriers for
transacylation (17 kcalmolꢀ1 for methylacrylate with CALB at
303 K based on the apparent kcat value given in Table 1). The
enzyme will thus encounter a constant concentration of s-cis/
s-trans acrylate conformers.
Table 1. Experimentally determined apparent kcat values when using
25 mm propan-1-ol as acyl acceptor in diisopropyl ether at 308C.
CALB[a] wt
CALB V190A
HiC[b]
RML[c]
[minꢀ1
2000
]
[minꢀ1
1000
]
[minꢀ1
]
[minꢀ1
]
1000
170
80
3000
40
510
60
240
170
520
A dihedral angle of 158 between the carbonyl and alkene
double bonds, which represents a small perturbation of the p
system, resulted in an energy increase of 0.3–0.4 kcalmolꢀ1
(Table S5). It was concluded that distortion of the p system is
unfavourable and that acrylates will more or less exclusively
bind in their s-cis or s-trans conformation. The path from such
a substrate–enzyme complex to a transition state will be very
important for the reaction rate. A substrate–enzyme complex
from which a minimal atomic motion is required and in which
interactions between the carbonyl oxygen and oxyanion hole
can help to break the p system of the substrate would be ben-
eficial.
50
250
280
120
[a] Candida antarctica lipase B. [b] Humicola insolens cutinase. [c] Rhizo-
mucor miehei lipase.
The presence of the a,b-double bond in methyl acrylate
leads to a reduction in apparent kcat of 4–75 times compared
to that of methyl propionate depending on the enzyme.
Methyl methacrylate reacted even more slowly with an addi-
tional reduction in apparent kcat of ten times for CALB wt.
Methyl a-chloroacrylate represents an activated compound
with increased reactivity compared to methyl methacrylate, in-
dicative of an electronic effect due to the fact that the chloro
and methyl groups are approximately isosteric. Interestingly,
Rhizomucor miehei lipase had the same apparent kcat for
methyl methacrylate and methyl acrylate.
In this work we investigated the relationship between acry-
late conformers bound to the active site and their path to the
transition state. We modelled the transition state as a tetrahe-
dral intermediate[10] and evaluated which conformer of the ac-
rylate it could be derived from with minimal atomic motion.
Figure 1 shows a modelled tetrahedral intermediate in CALB in
which the Og of the catalytic S105 and the sp3-hybridised car-
bonyl carbon of methyl methacrylate are covalently linked. A
minimised structure of the ES complex, which contains free
methyl methacrylate, is overlaid on the structure representing
the TS. The dihedral angle between the bond vectors connect-
ing the carbonyl oxygen, carbonyl carbon and the unsaturated
a- and b-carbon (marked in orange in Figure 1) in the cova-
lently bound substrate defines the conformation of the acry-
The distribution of substrate conformers for the different
acrylates was analysed by DFT B3LYP ab initio calculations with
the 6ꢀ311+G(2d,2p) basis set (Table 2). The geometry-opti-
mised structures were found to be either s-cis or s-trans con-
formers. The barrier to s-cis/s-trans isomerisation of methyl ac-
rylate was calculated to be 6 kcalmolꢀ1 at this level of theory.
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K. Hult and P.-O. Syrꢀn
tion of the dihedral angle defining the conformation of
enzyme-bound methyl acrylate and methyl a-chloroacrylate
was the same as for methyl methacrylate.
The molecular-dynamics simulations resulted in the distribu-
tion of acrylate dihedral angles in the TS for CALB-catalysed
transacylation shown in Figure 2. The range of possible dihe-
dral angles (from ꢀ1808 to 1808) was subdivided into intervals
of 158 in dihedral angle space. A dihedral angle of ꢀ158 to 158
corresponds to an apparent s-cis acrylate conformer whereas
ꢀ1658 to ꢀ1808 and 1658 to 1808 correspond to an apparent
s-trans conformer (in the tetrahedral transition state the p
system is broken, and thus the term “apparent” is used for the
acrylate conformer). We found that the abundance of popula-
tions with apparent s-cis and s-trans conformations was highly
dependent on the substrate and the catalyst. CALB wt had a
very low abundance of apparent s-cis conformers of methyl
methacrylate (1.3%) whereas the corresponding number was
17.7% for the more catalytically competent CALB V190A
mutant. Methyl acrylate had both apparent s-cis and s-trans
conformers in TS (15.5 and 51.1% abundance, respectively).
Thus the faster-reacting substrates and the more potent cata-
lyst displayed a higher abundance of apparent s-cis/s-trans
acrylate conformers in the TS model.
Figure 1. Tetrahedral intermediate, as a representation of the transition state
in the acylation of CALB with methyl methacrylate, overlaid on a minimised
structure of the ES complex containing free methyl methacrylate. Some
atoms are not shown for clarity. The dihedral angle defining the conforma-
tion of the covalently bound acrylate substrate (between the carbonyl
oxygen, carbonyl carbon and the unsaturated a- and b-carbons) is marked
in orange. Colour code: cyan=carbon, red=oxygen, blue=nitrogen,
white=hydrogen. The enzyme atoms in the ES-complex are coloured in
violet. The two structures were superimposed by using all enzyme atoms
with a resulting RMSD of 0.09 ꢂ.
late in the TS. Molecular dynamics simulations were used as a
tool to study the enzymatic preference for acrylate conforma-
tions in the TS, as described in the Experimental Section. Simu-
lations were performed on wt and mutants of CALB, Humicola
insolens cutinase (HiC) and Rhizomucor miehei lipase (RML)
with the three acrylate substrates discussed (methyl acrylate,
methyl methacrylate and methyl a-chloroacrylate). The defini-
We assumed that only populations with apparent s-cis/
s-trans acrylate conformers in the TS model were productive.
This assumption was based on the preference of the substrate
for being in a flat conformation. Furthermore, in the computed
Figure 2. Distribution of acrylate dihedral angles in the TS for enzyme-catalysed transacylation with CALB (the dihedral angle is between the bond vectors
connecting the carbonyl oxygen, carbonyl carbon and the a- and b-unsaturated carbon atoms in the enzyme-bound substrate). The dihedral angle space was
divided in 158 intervals. Populations with apparent s-cis or s-trans acrylate conformers (dihedral angles close to 08 and 1808, respectively) are marked in black.
The abundance of a certain population was based on the average value from several molecular-dynamics simulations with a minimum of 360 resulting struc-
tures.
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Rate of Enzymatic Acrylation
transition state for acylation of methyl acrylate at the DFT
B3LYP 6ꢀ31G(d,p) level of theory when using a minimal pro-
tein model consisting of a formate ion, an imidazole molecule
and a methanol molecule (for the catalytic triad) and two
methanol molecules (for the oxyanion hole), methyl acrylate
was found to be in a flat conformation (Figure S6).
Table 3. Relative abundances of populations with apparent s-cis and s-
trans acrylate conformations in the transition-state model and the corre-
sponding total relative probabilities of forming transition state for CALB.
The average abundances from several molecular dynamics simulations
are shown.[a]
Catalyst Substrate
Average
abundance
of s-cis
Average
abundance
of s-trans
Relative
probability
of forming
The relative abundance of productive s-cis/s-trans transition
states obtained from the molecular-dynamics simulation can
be translated into total relative probabilities of forming the
transition state for each catalyst–substrate pair if the abundan-
ces are normalised with the ground-state distribution of sub-
strate conformers. This is because an apparent s-cis acrylate
conformation in the transition state originates from an s-cis
ground-state substrate conformation. The relative final proba-
bility of forming a productive TS is thus given by the sum of
the s-cis/s-trans population abundance times the concentration
of the corresponding free ground-state acrylate conformer
(i.e., the concentration of the free s-cis or s-trans form). This
can be expressed as:
populations[b] populations[b] productive TS[c]
wt
0.013
0.177
0.202
0.155
0.478
0
0.003
0.048
0.117
0.251
0.277
V190A
wt
0
0
wt
0.511
0
V190A
Ptot ¼ ATS,s-cis ꢁ ½s-cisfreeꢂ þ ATS,s-trans ꢁ ½s-transfree
ꢂ
ð1Þ
Test of model
V190L[d]
0.003
0
0.001
Here Ptot is the total relative probability of forming a produc-
tive TS for the catalyst–substrate pair, ATS,s-cis and ATS,s-trans refer
to the abundance of productive s-cis/s-trans transition states
obtained from the molecular dynamics simulations, respective-
ly, and the terms within squared brackets refer to the concen-
trations of free substrate conformers. The result from these
calculations are shown in Table 3.
[a] Based on a minimum of 360 structures from the molecular-dynamics
simulation. We assumed that populations with apparent s-cis/s-trans acry-
late conformers were productive. The data are shown according to in-
creasing probability of forming the transition state. [b] The average abun-
dances were based on (from top to bottom): two simulations with a total
of 720 generated structures, three simulations with 1440 generated struc-
tures, one simulation with 720 structures, two simulations with 1440
structures, two simulations with 720 generated structures, one simulation
with 360 generated structures. [c] Corrected for the ground-state confor-
mational distribution of free acrylate as given in Table 2 and by using
Equation (1). [d] The V190L mutant with methyl methacrylate was used
for evaluation.
A good linear correlation between experimentally obtained
kcat values and the relative probabilities of forming transition
states was found, as shown in Figure S7. The slope of the
fitted line was found to be 1700 minꢀ1, and the intercept was
64 minꢀ1 with an R2 of 0.99. We tested this model by making
the CALB mutant V190L. This mutant was predicted to have a
low relative abundance of s-cis populations of methyl metha-
crylate in the TS by modelling. This was in agreement with a
measured apparent kcat of 50 minꢀ1.Clearly there is a prefer-
ence for acrylates to react in their low-energy s-cis or s-trans
ground-state conformations in enzymatic transacylation reac-
tions with CALB.
ductive s-cis/s-trans populations are given separately (see also
Table S7).
It could be concluded that productive populations had an
apparent s-cis or s-trans acrylate conformer in the TS (or a con-
formation very close to apparent s-cis/s-trans, Table S7). In
most cases, populations with apparent s-cis conformations
were found to have the lowest energy score. A good correla-
tion between the experimentally determined apparent kcat
values and the relative probability of forming productive tran-
sition states obtained from modelling and by evaluating the
energy score was found for CALB, as shown in Figure 3. Our
assumption about productive populations was thus concluded
to be fair for CALB.
The simple model for CALB based on assumed productive
conformations described above was refined by including the
interaction energy between enzyme and substrate in the tran-
sition state. The average force field interaction energy between
the acrylate substrate and enzyme in the TS was calculated for
each population of dihedral angles. Further, the average di-
hedral angle was used to account for deviations from apparent
s-cis and s-trans substrate conformations in the TS, bearing in
mind that the substrate in the transition state originated from
an sp2-hybridised precursor. The sum of the average interac-
tion energy and the cost of distorting the p system were used
to assign an energy score to each population (Supporting In-
formation). We assigned populations with productive transition
states to be those of lowest energy score or within 1 kcalmolꢀ1
of that value. The results of these calculations are shown in
Table 4 for wt and mutant CALB where the abundance of pro-
The correlation between experimental results and modelling
was also analysed for Humicola insolens cutinase and Rhizomu-
cor miehei lipase (Table S8). Productive populations were
chosen based on energy score and were translated into rela-
tive probabilities of forming transition states by correcting for
the ground-state distribution of acrylate conformers, as de-
scribed for CALB. For HiC, the correlation between experimen-
tal results and relative probabilities of forming productive tran-
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