Báo cáo khoa học: Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents potx

Modeling hydration mechanisms of enzymes in nonpolar
and polar organic solvents
Nuno M. Micae
ˆ
lo and Cla
´
udio M. Soares
Instituto de Tecnologia Quı
´
mica e Biolo
´
gica, Universidade Nova de Lisboa, Oeiras, Portugal
The ability of enzymes to work in media other than
water is now widely accepted, and is the basis of exten-
sive basic research on enzyme catalysis and many bio-
technological applications [1]. The fact that most
enzymes have evolved in an aqueous environment in
living cells does not mean that they cannot be trans-
ferred and be functional in a completely different kind
of medium [2–4]. Our recent molecular modeling stud-
ies have depicted the molecular mechanism of the
effects of different hydration percentages on the struc-
tural [5] and enantioselective properties of enzymes [6]
when placed in organic solvents such as hexane. Many
experimental studies in the field of nonaqueous enzy-
mology have focused their attention on demonstrating
that the amount of water in the organic medium plays
an important role in controlling the catalytic properties
of the enzymes [5–9]. These studies have shown that
when enzymes are used in organic solvents, water reta-
ins its fundamental role in controlling the physical

properties of the enzyme, and this role probably can-
not be taken by other solvent. In such systems, the
effect of water is complicated to investigate, because
this solvent is distributed in several phases; it can be in
the vapor phase, adsorbed to the support material, dis-
solved in the organic liquid phase, or bound to the
enzyme [10].
Of the total water added to the organic medium, the
effect of the organic solvents on the enzyme seems to
be primarily due to the water that is bound to the
enzyme [7,11]. This bound water is usually measured
experimentally in terms of the thermodynamic activity
of water, assuming that, for enzymatic reactions
Keywords
enzyme hydration; organic solvents; protein
modeling; water clusters
Correspondence
C. Soares, Instituto de Tecnologia Quı
´
mica
e Biolo
´
gica, Universidade Nova de Lisboa,
Av. da Repu
´
blica, Apartado 127, 2781-901
Oeiras, Portugal
Fax: +351 21 4433644
Tel: +351 21 4469610
E-mail:

Website: />(Received 21 December 2006, revised 1
March 2007, accepted 8 March 2007)
doi:10.1111/j.1742-4658.2007.05781.x
A comprehensive study of the hydration mechanism of an enzyme in non-
aqueous media was done using molecular dynamics simulations in five
organic solvents with different polarities, namely, hexane, 3-pentanone,
diisopropyl ether, ethanol, and acetonitrile. In these solvents, the serine
protease cutinase from Fusarium solani pisi was increasingly hydrated with
12 different hydration levels ranging from 5% to 100% (w ⁄ w) (weight of
water ⁄ weight of protein). The ability of organic solvents to ‘strip off’ water
from the enzyme surface was clearly dependent on the nature of the
organic solvent. The rmsd of the enzyme from the crystal structure was
shown to be lower at specific hydration levels, depending on the organic
solvent used. It was also shown that organic solvents determine the struc-
ture and dynamics of water at the enzyme surface. Nonpolar solvents
enhance the formation of large clusters of water that are tightly bound to
the enzyme, whereas water in polar organic solvents is fragmented in small
clusters loosely bound to the enzyme surface. Ions seem to play an import-
ant role in the stabilization of exposed charged residues, mainly at low
hydration levels. A common feature is found for the preferential localiza-
tion of water molecules at particular regions of the enzyme surface in all
organic solvents: water seems to be localized at equivalent regions of the
enzyme surface independently of the organic solvent employed.
Abbreviations
FF, force field; MD ⁄ MM, molecular dynamics ⁄ molecular mechanics; SPC, single point change.
2424 FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS
carried out in different media for a certain enzyme at
fixed water activity, the enzymes have equivalent
amounts of water bound [12]. This approach to expres-
sing water content in organic solvents has been a

standard in nonaqueous enzymology, simplifying the
interpretation and prediction of changes in enzyme
performance. It is commonly reported that the same
enzyme placed in different aqueous ⁄ organic mixtures
with the same water activity has similar catalytic con-
stants. This fact supports the idea that it is the
enzyme-bound water that modulates the catalytic
properties of the enzyme. However, this relationship
does not hold for polar solvents, especially at high
water activity [10]. It was hypothesized that solvents
such as alcohols are able to partially replace the role
of bound water, acting as ‘water replacers’ in promo-
ting enzyme activity [10]. The failure of water activity
to predict the critical amount of water needed for
enzyme activity suggests that the organic solvent also
has a role in modulating the structure and dynamics of
the enzyme, probably by taking part in the solvation
mechanism of the enzyme. It is clear that a concise
molecular picture of the solvation mechanism of
enzymes in nonaqueous solvents is needed.
Reviewing what is known about protein hydration
takes us back to early protein hydration studies on
dry proteins. There is a striking similarity between
the water-adsorption properties of proteins in air and
in organic solvents [13]. Early studies of Yang &
Rupley [14] and Rupley et al. [15], based on calori-
metric measurements of the heat capacity of the lyso-
zyme–water system, detailed the mechanism of the
hydration process of dry proteins. The authors [14]
pointed out that the hydration steps for lysozyme

resemble the Hill model for the localized adsorption
of adsorbate onto a heterogeneous surface [16]. They
interpreted the Hill model as follows: at low cover-
age, the adsorbate is dispersed on the surface; the
increase in adsorbate leads to the formation of a con-
densed phase of clusters; the clusters grow until the
surface is nearly covered and only the weakest sites
remain open; condensation of adsorbate over these
regions completes the adsorption process. With this
model, Yang proposed that water clusters can be
viewed as mobile arrangements centered on polar
regions of the protein surface that increase in size
and number as water is added. Protein–water adsorp-
tion isotherms in organic solvents and in the gas
phase [13] have shown that, at low water activity,
water adsorption by proteins suspended in nonpolar
organic solvents or by proteins equilibrated with
water from a gas phase are similar. This has led to
the conclusion that the presence of an organic solvent
has little effect on the interaction between the protein
and water in this water range.
Parker et al. [8] have detailed the mechanism of
enzyme hydration (using subtilisin Carlsberg) in non-
polar solvents using sensitive NMR experiments with
deuterated water. Their work clearly shows that in
nonpolar solvents (hexane, toluene, and benzene) water
is preferentially localized in the most polar regions of
the enzyme. The majority of the enzyme surface is in
direct contact with the organic solvent, and the forma-
tion of a monolayer of water over the protein surface

is thermodynamically unfavorable. However, no polar
organic solvent was used in this study. A similar study
[17], using water sorption isotherms of lysozyme in
nonpolar and polar organic solvents, previously sug-
gested the same mechanism of protein hydration. Io-
nizable sites are hydrated first, followed by polar and
nonpolar sites. However, when the sorption isotherms
of toluene and n-propanol in the same water activity
range are compared, they differ due to the competition
of the organic solvent for the enzyme hydration sites.
The hydration mechanism of enzymes in nonaque-
ous solvents seems to be dictated by many factors,
most of which have been addressed in the previous
cited reports. Not only do the properties of the organic
solvent determine the relative partition of water at the
enzyme surface at specific sites, but additionally, this
solvent has a significant role in the solvation mechan-
ism of the enzyme. In this context, it would be import-
ant for a molecular interpretation of the effects of the
different quantities of enzyme-bound water in non-
polar and polar organic solvents if the number of
water molecules boun d to the enzyme could be precisely
measured, characterized, and localized [10,18]. Our
work is a comprehensive study of the hydration mech-
anism of the enzyme cutinase in nonpolar (hexane, di-
isopropyl ether, 3-pentanone) and polar (ethanol,
acetonitrile) organic solvents with increasing hydration
levels. We have tried to determine how enzyme hydra-
tion occurs in these media, and what the role is of the
different organic solvents in the solvation mechanism

of the enzyme.
Results and Discussion
Enzyme structure
Protein molecular dynamics ⁄ molecular mechanics
(MD ⁄ MM) simulations in organic solvents have been
reported for several model systems [5,6,19–27]. These
simulation studies and the ones presented in this work
typically involve one suspended isolated single protein
molecule surrounded by water, ions, and organic
N. M. Micae
ˆ
lo and C. M. Soares Modeling hydration mechanisms of enzymes
FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS 2425
solvent. Real nonaqueous experimental studies are usu-
ally carried on an immobilization support; however,
the system would be more complex if other factors,
such as protein aggregation, played an important role
in the protein function. Our approach to the study of
protein structure and dynamics in such media has been
focused on understanding how proteins are affected by
the different hydration conditions when placed in
organic solvents [5,6]. These studies have shown that
the structure, dynamics and enantioselectivity of cu-
tinase in hexane can be optimized within a specific
water hydration range [5,6,27]. A more complete
understanding of the structural properties of this
enzyme in organic solvents is shown in Fig. 1. Cu-
tinase was simulated in five different organic solvents
of increasing dielectric constant with different hydra-
tion conditions. The water range studied for each

solvent is a key aspect in understanding how
the structural properties of the enzyme are modulated
by the hydration level. Testing different organic sol-
vents allows us to determine the role played by the
organic solvent in the stabilization ⁄ destabilization of
Fig. 1. Average rmsd of Ca atoms of cutinase from the X-ray structure in (A) hexane, (B) diisopropyl ether, (C) 3-pentanone, (D) ethanol, and
(E) acetonitrile, with different water percentages. Calculations of rmsd deviations were done in the 3–7 ns period for each simulation and for
all replicas. Error bars are estimated from the SE of five to seven replicate simulations.
Modeling hydration mechanisms of enzymes N. M. Micae
ˆ
lo and C. M. Soares
2426 FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS
the enzyme structure. The rmsd of the Ca atoms of
the protein fitted against the X-ray structure shows
that the enzyme structure is slightly different in each
organic solvent. There are low rmsd values for the
enzyme in ethanol, and high values in acetonitrile. For
hexane, diisopropyl ether, and 3-pentanone, the
enzyme Ca rmsd values at the different water percent-
ages are within 0.16 and 0.27 nm. How do these data
compare with experimental data? The molecular struc-
tures of several enzymes soaked in organic solvents
have been determined by crystallographic studies
[28–34], but only limited changes in protein structure
were detected as compared with conventional aqueous
crystals. Some authors argue that this approach could
hardly give any other answer, because if major con-
formational changes were to exist, it would be unlikely
that the crystal packing could be maintained [18].
Other methods are available that can provide struc-

tural information on these proteins in solution. CD
studies of a-chymotrypsin in organic solvents have
shown a clear correlation between water content and
secondary structure of the enzyme [9,35]. Fluorescence
measurements of enzymes in organic solvents have also
been used to investigate the structural changes of
enzymes. Kijima and coworkers [36,37] have shown
that a-chymotrypsin enantioselectivity and fluorescence
properties are correlated with the solvent composition.
These findings suggest a more dynamic picture of
enzyme structure rearrangement when enzymes are
placed in different organic solvents and have different
hydration levels. The enzyme Ca rmsd measurement
from our simulations at different water percentages
and organic solvents show that different solvation con-
ditions yield different enzyme structural properties.
This result is in agreement with the common experi-
mental observation that the solvent composition in
nonaqueous systems is able to affect enzyme structural
properties.
Further analysis of the rmsd (Fig. 1) suggests the
existence of minima in the rmsd data versus water per-
centage in the less polar organic solvents (hexane,
diisopropyl ether, and 3-pentanone). It is possible to
see that the structure of cutinase deviates less from the
X-ray structure when it is placed in hexane with 7.5%
water. In the case of cutinase in diisopropyl ether, we
obtained the lowest rmsd at 30% water. With a
slightly polar medium such as 3-pentanone, a local
rmsd minimum was observed at 40%; however, we

also observed low rmsd values at very low water per-
centages for this organic solvent. It can be seen that
the optimal water content that minimizes the difference
from the X-ray structure is displaced to higher water
levels as we increase the polarity of the organic
solvent, as seen experimentaly [38]. The dependence of
enzyme structural properties on different water con-
tents in organic solvents is a well-documented phenom-
enon observed for several enzymes. In some cases, a
bell-shaped behavior of structural and biocatalytic
properties is observed. The rmsd data of our model
enzyme placed in the less polar organic solvents with
different hydration levels resemble this type of bell-
shape behavior; this is clear in the case of hexane and
diisopropyl ether. However, this phenomenon is not
clearly seen in our simulations in the case of polar,
water-miscible organic solvents such as ethanol and
acetonitrile. This may be because only a small amount
of water is retained at the enzyme surface in the case
of polar organic solvents relative to nonpolar organic
solvents, as detailed below.
Water at the enzyme surface
Spatial probability density
A key aspect of this work is the analysis of the local-
ization of water at the enzyme surface. We have suc-
cessfully identified regions of high density of water in
close contact with the protein for the different organic
solvents tested. In Fig. 2, we show the spatial distribu-
tion probability of water at 25% water content for the
simulations in organic solvents and for the fully hydra-

ted simulation. This result was obtained by calculating
the water probability density from all configurations of
the last 3 ns for each organic solvent and for all repli-
cates. Probability densities were chosen in order to
drawn contours at percentiles approximately between
93% and 98% for hexane, diisopropyl ether, and
3-pentanone, and at 99% for ethanol, acetonitrile, and
water, giving the clearest picture for the preferential
hydration regions near the enzyme surface. From our
simulations, we see that water does not partition
appreciably to the organic solvent phase in the case of
hexane, diisopropyl ether, and 3-pentanone, whereas in
ethanol and acetonitrile, a considerable amount of
water is found in the organic phase (results not
shown). Note that in the fully hydrated system, we are
also able to identify regions of higher density of water
molecules.
The water arrangement over the enzyme surface
shows that there is no evidence of a complete water
monolayer covering the enzyme (in fact, a monolayer
would be possible at water percentages higher than
50%, although that never occurs), in accordance with
previous suggestions that this would be thermodynam-
ically unfavorable [8]. The water distribution at the
enzyme surface is clearly localized in certain regions,
leaving other parts of the enzyme surface in direct
N. M. Micae
ˆ
lo and C. M. Soares Modeling hydration mechanisms of enzymes
FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS 2427

contact with the organic solvent. This clearly suggests
that the role of the organic solvents should not be
undervalued, given that a significant enzyme surface
area is solvated by the organic solvent in all organic
solvents tested. This means that water per se might not
account entirely for the solvation process of the
enzyme.
An important discussion point that arises from our
studies is that the preferential binding sites of water at
the enzyme surface seem to be independent of the
organic solvent. This is supported by the observation
that the water spatial probability distributions seem to
be equivalent for the same enzyme, regardless of the
organic solvent used (Fig. 2). This appears to be true if
we look at Fig. 2 and try to compare the spatial prob-
ability densities of water in hexane, diisopropyl ether,
and 3-pentanone. They show that water is distributed
over similar regions of the enzyme in these three sol-
vents. In the case of more polar solvents such as ethanol
and acetonitrile, we see that the water in these systems
is found in regions also present in the nonpolar solvents.
As the enzyme surface does not change dramatically
when the enzyme is placed in different organic solvents,
water molecules seem to populate equivalent sites that
correspond to the areas of exposed charged ⁄ polar side
chains hydrated to a higher or lower degree according
to the polarity of the organic solvent.
Nonpolar organic solvents
In order to obtain a more precise picture of water at
the surface of the protein, we looked at the number of

water molecules within a specific layer of 0.25 nm from
the surface of the protein (Fig. 3), and the ratio of
Fig. 2. Spatial distribution probability density
of water in (A) hexane, (B) diisopropyl ether,
(C) 3-pentanone, (D) ethanol and (E) aceto-
nitrile with 25% water and (F) in the fully
hydrated system. The molecular surface
corresponds to the average structure of cu-
tinase from the 3–7 ns period, for each sol-
vation system and for all replicas. For each
organic solvent, two sides of the enzyme
are shown in order to give a complete view
of the surface. Each view of the enzyme
has the same orientation in all organic sol-
vents. The contours enclose regions with a
probability density above 9 · 10
)6
A
˚
)3
for
hexane, diisopropyl ether, and 3-pentanone,
4 · 10
)6
A
˚
)3
for ethanol, 3 · 10
)6
A

˚
)3
for
acetonitrile, and 1 · 10
)6
A
˚
)3
for the fully
hydrated system.
Fig. 3. Average number of water molecules less than 0.25 nm
away from the enzyme surface, for each organic solvent and water
percentage. Error bars are estimated from the SE of five to seven
replicate simulations. The total number of water molecules corres-
ponding to each hydration level, for comparison with the number
bound, is given in supplementary Table S3.
Modeling hydration mechanisms of enzymes N. M. Micae
ˆ
lo and C. M. Soares
2428 FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS
water to organic molecules in the region beyond this
layer (supplementary Table S1), for all organic solvents
and hydration levels. This criterion is intended to cap-
ture the first layer of water molecules in direct contact
with the enzyme. The first evidence from these curves
is the fact that they resemble the shape of the water
adsorption isotherms of enzymes in nonaqueous sol-
vents [13,17,39,40], although in these experimental
reports, water adsorption is plotted as a function of
water activity rather than water content. These curves

show, first, a rapid increase in bound water, followed
by a second step in which there is a slow increase, and
then a third step of high water activity, where again
there is a sharp increase. The water range that we tes-
ted seems to comprise the two initial steps. Note that
the curves can easily discriminate nonpolar and polar
solvents. In nonpolar solvents, water is highly retained
at the enzyme surface, whereas in polar solvents, water
is only weakly retained.
In the particular case of hexane, most of the water
is located in this first hydration shell around the
enzyme, covering a large proportion of the surface
area of the enzyme but not achieving full coverage, as
seen in the previous section. This organic solvent is the
one that allows the retention of the highest amount of
water at the enzyme surface. The remaining water that
is not in direct contact with the enzyme is found in
secondary hydration layers. Changing to a slightly
polar solvent such as diisopropyl ether, we see the
same trend for the water amount curve, but in this
case the amount of water in direct contact with the
enzyme is slightly lower, and it decreases even more as
the polarity of the organic solvent increases, as seen
for 3-pentanone. This clearly suggests that, to obtain
the same amount of water bound to the enzyme, we
need to add more water to the system as we move to
more polar solvents. The general trend for the amount
of water at the enzyme surface observed for these three
solvents shows that, at low water percentages, most of
the water present in the system is found at the surface

of the enzyme, and as water is added, it expands its
coverage over the enzyme surface up to a certain limit.
For instance, it is possible to see that in diisopropyl
ether, the hydration of the enzyme surface reaches a
saturation point at about 40% water, corresponding to
90 water molecules at a distance less than 0.25 nm
from the enzyme surface. For the case of 3-pentanone,
we see that at water percentages above 60%, corres-
ponding to 80 water molecules, there is almost no
more water retained at the enzyme surface. In general,
it can be seen that as we change from apolar solvents
(hexane) to a slightly polar organic solvent (3-penta-
none), it becomes energetically favorable to have
organic solvent molecules instead of water molecules
solvating the enzyme.
Polar organic solvents
In water-miscible organic solvents such as ethanol and
acetonitrile, the competition between the organic sol-
vent and water for the enzyme surface is higher. These
two organic solvents can mimic the nonbonding prop-
erties of water, and can effectively compete for the
polar regions of the enzyme. This is clearly seen in
Fig. 3, which shows that the amount of water bound
at the enzyme surface is very low as compared to the
situation with nonpolar solvents. In polar organic sol-
vents, the amount of water bound to the enzyme
increases slowly as water is introduced to the system,
showing that, to obtain the same amount of water
bound to the enzyme in nonpolar solvents such as hex-
ane, it is necessary to add very high amounts of water.

The amount of organic solvent bound to the enzyme is
very high, and the solvent clearly acts as a water re-
placer in many polar and nonpolar regions of the
enzyme surface. This phenomenon clearly correlates
with previous observations that polar organic solvents
strip water from the enzyme surface to a higher extent
than nonpolar ones [41]. This result is also in agree-
ment with the observation of McMinn et al. [17] show-
ing that in polar organic solvents, the amount of water
bound to the enzyme at high water activities is signifi-
cantly lower relative to the case when nonpolar
organic solvents are used. In this case, the organic sol-
vent has a significant role in solvating the enzyme, and
also takes part in the modulation of the structural and
dynamic properties of the enzyme. It is also of note
that ethanol and acetonitrile are significantly different
in their ability to strip off water from the enzyme.
In ethanol, at almost all hydration levels, we find twice
as much water bound to the enzyme as in aceto-
nitrile; however, experiments show that more water is
bound when the enzyme is equilibrated with a given
water concentration in acetonitrile as compared with
ethanol [17].
Water structure
In our simulated systems, we see that water molecules
can be found in the bulk organic phase or at the
enzyme surface. In the water-miscible organic solvents,
we see a considerable amount of water in the organic
solvent phase (results not shown). However, in all
organic solvents, water seems to be organized in clus-

ters at the enzyme surface (Fig. 4A). These water
clusters might have a substantial functional role in
N. M. Micae
ˆ
lo and C. M. Soares Modeling hydration mechanisms of enzymes
FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS 2429
modulating the structural and dynamic properties of
the enzyme, as suggested previously.
In Fig. 4A, we show the number of water clusters at
the enzyme surface. A cluster is defined by two or
more water molecules at a minimum distance of
0.3 nm (distance between oxygen atoms). Note that
this criterion might not account for single water mole-
cules interacting with polar residues or water molecules
solvating the ions in solutions (as oxygen atoms in this
case might be more than 0.3 nm distant). In Fig. 4B,
we show the average number of water molecules per
cluster. Again, the behavior in nonpolar and polar sol-
vents is easily distinguishable by this property. In the
presence of nonpolar organic solvents, the number of
water clusters is almost identical in all hydration con-
ditions up to 25% (Fig. 4A). In the nonpolar organic
solvents, the number of clusters grows rapidly as water
is added, indicating that water is being organized in
clusters of two or more water molecules at the enzyme
surface, hydrating specific spots of the enzyme. The
number of clusters increases up to 25% water content,
and beyond this water level, the number of clusters
remains constant. This indicates that, as water is added
to the system, new clusters of water are formed at

available specific regions at the enzyme surface, which
become fully occupied at 25% water content. The size
of the clusters also grows as water is added, meaning
that the water that is gradually introduced is also dis-
tributed on pre-existing water clusters. Water clusters
in nonpolar solvents are of similar size at low water
percentages, but as water is added, the clusters in hex-
ane become significantly larger than those in diisopro-
pyl ether and 3-pentanone (Fig. 4B).
In polar organic solvents, the water organization at
the enzyme surface is different from that in the nonpo-
lar organic solvents. The cluster size increases more
slowly as water is added, suggesting that most of the
water introduced into the system is preferentially
localized in the bulk organic solution. The size of the
clusters in ethanol and acetonitrile is fairly identical up
to 50% water content. Above this level, water clusters
in ethanol are slightly larger that those in acetonitrile.
The molecular picture that arises from this analysis
of polar organic solvents is that water is largely frag-
mented into single water molecules and small clusters
of water molecules around the protein. In the case of
nonpolar solvents, water is tightly bound to the
enzyme and organized in clusters that grow in number
and size in proportion to the water added.
Ions in nonaqueous systems
Ions play an important role in nonaqueous media, as
they will allow the neutralization of exposed charged
residues of the enzyme that cannot form intramolecu-
lar ion pairs [42]. This is evident from the X-ray struc-

ture of trypsin in cyclohexane, which shows the
existence of sulfate ions forming salt bridges or hydro-
gen bonds with residues or water molecules [34]. Work
with different ion concentrations in nonaqueous system
has also shown that ions have a marked impact on
enzyme activity, relative to enzymes with no added salt
[43–45].
In all organic solvent simulations, we have 10 Na
+
and 10 Cl
–
docked to cutinase, as described in a previ-
ous study [5]. These ions neutralize individual charged
groups at the enzyme surface. We have seen that at
equilibrium, water rearranges itself around the protein
according to the type of the organic solvent used and
the amount of water present in the system. The same
seems to be true for the ions. In Fig. 5, we analyze
Fig. 4. (A) Average number of water clusters for each organic sol-
vent and water percentage. (B) Average size of the water clusters
shown in (A). Error bars are estimated from the SE of five to seven
replicate simulations. See text for details and comments regarding
this analysis.
Modeling hydration mechanisms of enzymes N. M. Micae
ˆ
lo and C. M. Soares
2430 FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS
how many charged residues are neutralized by sodium
(Fig. 5A) and chloride (Fig. 5B) counterions. The first
evidence is that ions are not irreversibly bound to the

enzyme, as some charged residues initially stabilized by
ions are preferentially compensated by water mole-
cules, particularly at high water percentages. Sodium
ions bound to the enzyme seem to be highly conserved
at low water percentages in all organic solvents. As we
add water, sodium ions seem to be displaced from the
enzyme surface to the same extent in all organic sol-
vents. These ions are found free in solution, hydrated
by water molecules, which, as we have seen, are organ-
ized in clusters. Another common observation is the
formation of Na
+
Cl
–
ion pairs and, more rarely,
Na
+
Cl
–
tetrads in solution or at the enzyme surface,
where one of the negative or positive pair is a charged
residue. Chloride ions are also displaced from the
enzyme surface by water in the same way as sodium
ions. However it seems that more polar solvents such
as ethanol and acetonitrile, even at low water percent-
ages, are able to replace chloride ions at the enzyme
surface. This phenomenon is more evident in acetonit-
rile, as these chloride ions are rapidly removed from
the enzyme, even at very low levels of hydration. At
high levels of hydration, only one ion or even none is

found bound to the enzyme. Another phenomenon
seen in Fig. 5A at low water percentages in nonpolar
solvents is that some of the chloride ions stabilize more
than one positive charged residue, as the 10 Cl
–
are in
the proximity of more than 10 positive charged resi-
dues. What these results suggest is that ions can also
be ‘stripped off’ from the enzyme surface by the water
molecules. The charged residues initially stabilized by
ions at low water percentages become preferentially
compensated by water molecules as the water content
increases. This suggests that at low hydration levels,
ions are important in the stabilization of charged resi-
dues, but as the system becomes ‘more aqueous’, the
exposed charged residues are preferentially stabilized
by water molecules. The loss of the charge-counteract-
ing effect provided by the ions near exposed charged
residues could also be responsible for the structural
changes observed at higher water percentages in all
organic solvents.
Water dynamics
We have seen that organic solvents with increasing
polarity can structure, in different ways, the water at
the enzyme surface. It is also important to question
how the dynamics of the water are modulated by the
presence of the different organic solvents. In a recent
NMR study [46], the authors suggested a hydration
model of subtilisin in tetrahydrofuran that comprised
tightly bound, loosely bound and free water. To ana-

lyze the water dynamics at the enzyme surface, we
recorded all the hydration events of all water mole-
cules in the system. A hydration event is the total time
for which one water molecule is inside a layer of
0.25 nm around the enzyme surface. The analysis was
done during the last 3 ns of all trajectories for water
contents of 25% and 60%. All hydration events of all
water molecules for a specific water content and
organic solvent are collected and grouped in a fre-
quency histogram. For a clear analysis, the data were
fitted using the Levenberg–Marquardt method to a
two-exponential equation of the form:
f ðxÞ¼a þ b  e
ÀcÂx
þ d  e
ÀfÂx
where x stands for the time in ps that a water molecule
is inside a layer of 0.25 nm around the enzyme surface,
and f(x) is the frequency of occurrence of that period
Fig. 5. Average number of (A) negatively and (B) positively charged
residues neutralized by sodium and chloride ions, respectively, for
each organic solvent and water percentage. Error bars are estima-
ted from the SE of five to seven replicate simulations.
N. M. Micae
ˆ
lo and C. M. Soares Modeling hydration mechanisms of enzymes
FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS 2431
of time. Final parameters and standard errors are
shown in supplementary Table S2.
The residence times of the water molecules at the

enzyme surface at 25% and 60% water content in the
different organic solvents and in the fully hydrated sys-
tem are shown on Fig. 6. A general overview of this
figure indicates that many of the water molecules have
very low residence times at the enzyme surface. How-
ever, a significant proportion of the water is retained
at the enzyme surface on a nanosecond time scale in
all organic solvents. At 25% water content (Fig. 6A),
it is possible to distinguish the effects of nonpolar and
polar organic solvents on the dynamics of the water at
the enzyme surface. Water molecules in nonpolar sol-
vents are retained for longer periods of time at the sur-
face of the enzyme than in polar organic solvents.
Particularly at 25% water content, the water residence
times in ethanol and acetonitrile are equivalent to
those in the pure aqueous system. At 60% water con-
tent (Fig. 6B), all four organic solvents (no data for
hexane at this water content) modulate the hydration
events in a progressive way; that is, water is retained
for less time at the enzyme surface as the polarity of
the organic solvent increases.
It seems that, besides the differential structuring of
water by the organic solvent, these water molecules
organized in clusters at the enzyme surface do not
behave as in a bulk aqueous solution. Their dynamic
properties, with respect to the residence time at the
enzyme surface, are modulated by the polarity of the
organic solvent.
Concluding remarks
We have performed a systematic simulation study of

the hydration mechanism of one enzyme in three dis-
tinct classes of solvent: nonpolar organic solvents,
polar organic solvents, and water. We consider the
effect of five different organic solvents, with different
water percentages, on the structural properties of one
model enzyme. It is shown that the structural proper-
ties of the enzyme in the less polar solvents (hexane,
diisopropyl ether, and 3-pentanone) give a bell-shape
curve, indicating that there is an optimum hydration
level that allows the existence of a native-like structure
in solution. This optimal hydration level for each
organic solvent is obtained at increasing water percent-
ages as we move to more polar solvents. Our study
also provides a detailed molecular picture of the
hydration mechanism in the organic media, as indica-
ted previously by experimental findings obtained by
hydration studies in organic solvents by NMR and
also from water adsorption experiments. Our results
show that water in nonaqueous media is organized at
the enzyme surface in clusters of water molecules
hydrating preferentially charged ⁄ polar residues. These
clusters populate identical enzyme surface regions
when the enzyme is placed in different organic sol-
vents. As water is added, these clusters grow in num-
ber and size. The nature of the organic solvent is able
to determine the size and number of clusters. Nonpolar
solvents allow the existence of large clusters of water
molecules at the enzyme surface, whereas polar sol-
vents fragment these clusters into smaller aggregates.
Polar solvents have the ability to replace water at some

enzyme surface regions and contribute effectively to
the structure and dynamics of the enzyme. This means
that water activity per se may not be sufficient to char-
acterize the solvation of enzymes, and thus, water
activity values in different organic solvent might not
correlate directly with catalytic properties measured
experimentally. Ions seem to be preferentially bound
to the enzyme at low hydration levels. Owing to the
presence of the organic solvent, water is retained for
A
B
Fig. 6. Water residence time frequency for each organic solvent
with (A) 25% water and (B) 60% water. See text for details and
comments regarding this analysis.
Modeling hydration mechanisms of enzymes N. M. Micae
ˆ
lo and C. M. Soares
2432 FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS
longer at the enzyme surface, and this is more evident
in solvents with very low polarity. On the other hand,
in high-polarity solvents, water at the enzyme surface
behaves similarly as in the fully hydrated system. This
study has provided a detailed molecular picture of the
hydration mechanism of an enzyme, and shown it to
be clearly dependent on the nature of the organic sol-
vent and water content.
Experimental procedures
Organic solvents force field
The organic solvents employed in this study were: hexane,
diisopropyl ether, 3-pentanone, ethanol, and acetonitrile.

These organic solvents are commonly used in nonaqueous
enzymology studies, and are parameterized for MD ⁄ MM
simulations. Hexane was modeled as a flexible united atom
model using the gromos96 43a1 alkane parameters [47];
diisopropyl ether was taken from Stubbs et al. [48] and
adapted to the gromos96 43a1 force-field (FF) [49,50];
3-pentanone is found in the gromos96 43a1 FF [51];
ethanol is also present in the gromos96 43a1 FF, and a
new parameterization of acetonitrile [52] was recently done
for this FF.
These organic solvents have increasing dielectric proper-
ties and different partition coefficients [53] (Table 1). The
rationale for the choice of these organic solvents was to
have two groups with distinct properties, those that are
immiscible with water and that have low polar characteris-
tics (hexane, diisopropyl ether, and 3-pentanone), and those
that have polar properties and are water miscible (ethanol
and acetonitrile).
System setup
The general simulation methodology applied in the
MD ⁄ MM simulations of cutinase in nonaqueous solvents
with increasing amounts of water was similar to the one
that we applied in a previous study [6]. The 1.0 A
˚
resolu-
tion cutinase structure of Longhi was used [54], and the
protonated state of charged residues was estimated using a
methodology described previously [55]. The selection of
counterion positions and the different amounts of water
hydrating the enzyme was done as explained in detail else-

where [5]. Five to seven replicates of 12 hydration levels
were chosen, ranging from 5% to 100%; see supplementary
Table S3 for a complete description of the molecular com-
position of each system. The replicates are composed of
equivalent molecular systems (enzyme, ions, water, and
organic solvent) with water molecules at slightly different
positions at the enzyme surface. Different hydration ranges
were chosen for each organic solvent according to the
knowledge that the critical amount of water that optimizes
the structural and dynamic properties of the enzyme
depends on the polarity of the organic solvent used [7].
Each replicate of cutinase hydrated with a specific amount
of water was placed in a dodecahedral box with a minimum
distance between the protein and box wall of 0.8 nm, and
solvated with an equilibrated configuration of organic sol-
vent molecules at 300 K. Three replicate simulations of
cutinase with ions in a fully hydrated system with single
point change (SPC) water were also done.
MD ⁄ MM simulations
MD ⁄ MM simulations were performed with the gromacs
package [56,57] using the gromos96 43a1 FF [49,50]. Bond
lengths of the solute and organic solvent molecules were
constrained with lincs [58], and those of water with settle
[59]. Nonbonded interactions were calculated using a twin-
range method [50] with short-range and long-range cut-offs
of 8 A
˚
and 14 A
˚
, respectively. The SPC water model [60]

was used in aqueous and in nonaqueous simulations. A
reaction field correction for electrostatic interactions [61,62]
was applied, taking a dielectric of 54 for the fully hydrated
system with SPC water [63]. For the nonaqueous systems,
the dielectric constant was chosen according to the experi-
mental value reported in Table 1. The simulations were
started in the canonical ensemble with initial velocities from
a Maxwell–Boltzmmann distribution at 300 K, and run
for 50 ps with position restraints applied to all heavy atoms
of the protein and water molecules (force constant of
10
6
kJÆmol
)1
Ænm
)2
) and a temperature coupling constant of
0.01 ps, allowing the equilibration of the organic solvent. A
further 50 ps of restrained simulation with the same force
constant on the protein heavy atoms and temperature coup-
ling constant was done for the equilibration of water mole-
cules. A final step of 50 ps was done with restraints only
applied to the Ca carbons of the enzyme and a temperature
coupling constant of 0.1 ps. The unrestrained simulations
were done in the isothermal–isobaric ensemble with an
integration time step of 2 femtoseconds. The protein, ions,
organic solvent and water were coupled to four separated
heat baths [64] with temperature coupling constants of
0.1 ps and a reference temperature of 300 K. The pressure
control [64] was implemented with a reference pressure of

Table 1. Dielectric constant and partition coefficient (log P) [53] of
the organic solvents employed in this work.
Dielectric constant
(temperature K)
Partition coefficient
(log P)
Hexane 1.9 (293.2) 4.00
Diisopropyl ether 3.8 (303.2) 1.52
3-Pentanone 17.0 (293.2) 0.82
Ethanol 25.3 (293.2) ) 0.30
Acetonitrile 36.6 (293.2) ) 0.54
N. M. Micae
ˆ
lo and C. M. Soares Modeling hydration mechanisms of enzymes
FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS 2433
1 atm and relaxation times of 0.5 ps and 1.3 ps, for water
or organic ⁄ water solvent simulations, respectively.
Acknowledgements
The authors acknowledge helpful discussions with Dr
Anto
´
nio M. Baptista and Professor Susana Barreiros,
and financial support from Fundac¸ a
˜
o para a Cieˆ ncia e
a Tecnologia, Portugal, through grants POCTI ⁄ BIO ⁄
57193 ⁄ 04 and SFRH ⁄ BD ⁄ 10611 ⁄ 2002.
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Supplementary material
The following supplementary material is available

online:
Table S1. Ratio of water to organic molecules in the
region away from the protein (beyond 0.25 nm from
the enzyme surface).
Table S2. Parameters and SEs of the water residence
time fitted data from Fig. 6.
Table S3. Number of water molecules, organic solvent
and NaCl present in the molecular system.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-
ponding author for the article.
Modeling hydration mechanisms of enzymes N. M. Micae
ˆ
lo and C. M. Soares
2436 FEBS Journal 274 (2007) 2424–2436 ª 2007 The Authors Journal compilation ª 2007 FEBS