α-Chymotrypsin superactivity in quaternary ammonium salt solution: Kinetic and computational studies

Article abstract of DOI:10.1039/c6ra07425a

As previously reported, quaternary ammonium salts with bulky hydrophobic portions provoke a superactivation of α-chymotrypsin in aqueous solution: this is the case of the surfactant cetyltributylammonium bromide (CTBABr) and the corresponding salt tetrabu

Full text of DOI:10.1039/c6ra07425a

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a-Chymotrypsin superactivity in quaternary
ammonium salt solution: kinetic and
Cite this: RSC Adv., 2016, 6, 46202
computational studies
a
Laura De Matteis,† Francesca Di Renzo,a Raimondo Germani,b Laura Goracci,c
*
Nicoletta Spretia and Matteo Tieccob
As previously reported, quaternary ammonium salts with bulky hydrophobic portions provoke a superactivation
of a-chymotrypsin in aqueous solution: this is the case of the surfactant cetyltributylammonium bromide
(CTBABr) and the corresponding salt tetrabutylammonium bromide (TBABr). In order to achieve a broader
knowledge of the enzyme–additive interactions, in this paper the activity and stability of a-chymotrypsin
were tested in the presence of additives with slightly modified bulky ammonium groups. The effect of three
additives with
a
benzylic group as substituent (benzyltrimethylammonium bromide (BzTMABr),
benzyltributylammonium bromide (BzTBABr) and benzyldodecyldimethylammonium bromide (BzDDABr)) was
investigated. A significant increase in instantaneous activity, but a deactivation of enzyme, faster than in pure
buffer, was observed. Moreover, two novel dicationic salts, (1,8-bis(tributylammonium)octane dibromide
(bisBOAB) and 1,4-bis(tributylammonium)xylene dibromide (bisBAB)) were designed and synthesized in order
to evaluate the effect of two tributylammonium head groups with a different spacer. BisBOAB provoked
superactivation and stabilization effects in
a way similar to the “homologue” TBABr, but at lower
concentration. In contrast, when the benzyl group was constrained within the spacer structure, the obtained
superactivity was lower than in the presence of a more flexible hydrocarbon chain spacer, and enzyme
deactivation was faster. Molecular modelling studies allowed us to rationalize the hypotheses derived from
kinetic evidence. The results confirmed that the improvement in the catalytic properties observed in the
presence of additives with a bulky, hydrophobic ammonium head group could be addressed to an increase
in the overall hydrophobicity of the a-chymotrypsin catalytic site.
Received 21st March 2016
Accepted 2nd May 2016
DOI: 10.1039/c6ra07425a
www.rsc.org/advances
acid residues. These interactions seem to cause the unfolding of
the enzyme tertiary structure and the loss of its activity.
Introduction
The presence of surfactants and other additives can also
produce conformational changes in the enzyme that allow
achieving better catalytic properties. In such cases, functional
groups of additives play a relevant role in these effects, since any
selective interaction may depend on the structures of both
enzyme and the additive.
Furthermore, in aqueous solutions the surfactant can exist
as monomers (below the critical micelle concentration, CMC) or
as micellar aggregates (above the CMC); so the effect of
surfactant on enzyme structure and catalytic properties also
depends on its concentration.
The effect of surfactants on enzymatic performance in aqueous
solutions has been widely investigated and reviewed.1–4 Different
techniques have been used to study protein–surfactant interac-
tions and the mechanism of protein denaturation by sodium
dodecyl sulfate (SDS) has been studied in many different works.
The nature of the interaction has been reported to be both elec-
trostatic and hydrophobic. An initial electrostatic binding occurs
between the charged head group of the surfactant and ionic sites
on the protein surface; this is followed by lipophilic interactions
between the alkyl chain of the surfactant and hydrophobic amino
The effect of additives on enzymatic activity has been also
studied in many relevant works in literature concerning cova-
lent binding of amphiphiles to enzymes. Better catalytic prop-
erties of the conjugates were observed both in aqueous
solutions5 and in organic solvents,6,7 increasing the relevance of
using amphiphilic additives in this topic.
a-Chymotrypsin (EC 3.4.21.1) is a globular serine protease
that has been widely studied in aqueous solutions, since its
structure and mechanism of action are well known.8–10 Thus it
aDepartment of Physical and Chemical Sciences, University of L'Aquila, Via Vetoio,
I-67100 Coppito, L'Aquila, Italy. E-mail: lauradem@unizar.es
bCEMIN, Centre of Excellence on Nanostructured Innovative Materials, Department of
Chemistry, Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, I-
06123 Perugia, Italy
cLaboratory for Chemoinformatics and Molecular Modelling, Department of
Chemistry, Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, I-
06123 Perugia, Italy
´
† Current address: Instituto de Nanociencia de Aragon (INA), Universidad de
Zaragoza, C/Mariano Esquillor s/n, 50018 Zaragoza, Spain.
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was selected as a model enzyme to investigate the effects of CMC. Moreover, the curve of reaction rate versus surfactant
differently structured additives on its catalytic properties. concentration was bell-shaped. At low surfactant concentrations,
Recently biological buffers, polyelectrolytes and polyamines the presence of few CTBABr molecules can induce enzyme
were used to improve the catalytic activity of a-chymotrypsin (a- superactivity, while the reduction of reaction rate with increasing
CT); favorable electrostatic interactions between the additive surfactant concentration was attributed to the partition of the
and the enzyme, or substrate, seemed to contribute to the substrate in the micellar aggregates. These results proved that an
superactivation of a-CT.11–13
Many works in the literature dealt with the effect of surfac- responsible for a-CT superactivity.
increase in the size of the trialkylammonium head group was
tants on a-CT activity; in one of the early papers the inhibiting
The positive effect of a bulky head group was also observed
effect of several ionic and neutral surfactants on enzyme activity for the hydrolysis of p-nitrophenyl acetate (PNPA) in solutions of
is reported.14 In our previous studies on the effect of cationic ammonium and phosphonium surfactants.17–19 The highest
surfactants on a-CT-catalyzed hydrolysis of N-glutaryl-L-phenyl- values of superactivities were observed with dodecyl-
alanine-p-nitroanilide (GPNA) we showed that not only the charge triphenylphosphonium bromide and cetyltributylphosphonium
of the surfactant affects enzyme activity, but also the size of the bromide and such effects were due to an increase in the catalytic
head group has relevance.15,16 In particular, among cationic rate constant (kcat).
cetyltrialkylammonium bromide surfactants, cetyltrimethyl-
Based on the previously reported positive inuence observed
ammonium bromide (CTABr) showed a deactivating effect on the for CTBABr on a-CT activity, the effect of tetrabutylammonium
enzyme. On the contrary, it was reported that the instantaneous bromide (TBABr) was also studied.20 This organic salt has the
activity was signicantly favored in the presence of cetyl- same head group of CTBABr but it is unable to form micelles
tributylammonium bromide (CTBABr) both below and above the because of the lack of the hydrophobic chain. Enzyme activity
Fig. 1 Structures of the additives used in this work.
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showed similar effects on hydrolysis rate to those obtained with and 1,4-bis(tributylammonium)xylene dibromide (bisBAB) were
CTBABr, conrming the activating role of a bulky, hydrophobic, employed to study their effect on enzyme activity and stability.
ammonium group.
These molecules have two tributylammonium head groups, con-
Different authors developed a different strategy to change the strained by a spacer of different exibility. Structures and acro-
surfactant hydrophobic/hydrophilic balance, without altering the nyms of all the additives employed in this paper are reported in
characteristics of the micellar interface, by modifying the Fig. 1.
hydrocarbon chain length.21,22 Unfortunately, their results did not
Finally, molecular modelling studies were performed to
agree with each other. The hydrolytic activity of a-CT was studied evaluate the most favorable binding poses of the additives in the
in the presence of alkyldimethylethanolammonium bromide enzyme catalytic site in the attempt to rationalize results
surfactants with different chain length (from C-10 to C-16) at derived from kinetic studies.
concentration below and above the CMC. The kcat increased with
the surfactant chain length, conrming that a surfactant with
higher hydrophobicity induces enzyme superactivity.21 An
Experimental
Crystalline bovine pancreatic a-chymotrypsin (EC 3.4.21.1) (a-
CT, 24.8 kDa, type II: 3 times crystallized, dialyzed and lyophi-
lized) and the substrate N-glutaryl-L-phenylalanine-p-nitro-
anilide (GPNA) are purchased from Sigma. Buffered solutions of
enzyme and substrate were freshly prepared immediately before
use. The chemical Tris used for buffer preparation and p-
nitroaniline used for molar absorption coefficient determina-
tions are from Aldrich. The commercial grade salts benzyl-
tributylammonium bromide (BzTBABr) and benzyltrimethyl-
ammonium bromide (BzTMABr) and dodecylbenzyldimethy-
lammonium bromide (DDBzABr), are from Sigma-Aldrich and
puried by crystallization; synthetic 1,8-bis(tributylammonium)
octane dibromide (bisBOAB) and 1,4-bis(tributylammonium)
xylene dibromide (bisBAB) have been prepared in our labora-
tories by direct quaternization from respective dibromides, i.e.
1,8-dibromooctane and a,a0-dibromo-p-xylene, with tributyl-
amine in slight excess. Both the additives are puried and
characterized.
increase in a-CT activity was also observed in presence of three
alkyltrimethylammonium bromide surfactants (CnTABr) with
different chain lengths (n ¼ 12, 14, 16). The maximum catalytic
efficiency was registered at concentrations closer to surfactant
CMC. However, in this case, the increase in surfactant chain
length led to a lower superactivity.22
The characteristics of the substrate are also important in
determining the effect of surfactants on a-CT hydrolysis
rate.23–25 In the presence of the same surfactant and at the same
concentration (above the CMC), the distribution of the
substrate between aqueous and micellar pseudophase strongly
depends on its shape, charge and hydrophobicity. Therefore,
the amount of free substrate available for catalysis, and then the
kinetic behavior of the enzyme, could be very different.
Recently, Valiullina and co-workers carried out a study on a-CT
catalyzed hydrolysis of N-benzoyl-L-tyrosine-p-nitroanilide (BTNA)
in aqueous solutions of gemini surfactants, reporting that the
length of both the alkyl chain and the polymethylene spacer
affects the enzyme activity.26,27 In particular, it was reported that
the hydrolysis rate increased in the presence of surfactant with
shorter alkyl chain (C-10 and C-12), while reaction was inhibited
with additives with longer hydrocarbon radicals. These effects are
closely related to surfactant concentration. Monomers in fact
positively interacted with the enzyme improving its catalytic
activity; on the other hand, the increase of micellized surfactant
reduced the amount of free substrate, decreasing the hydrolysis
rate. Moreover, a-CT activity was affected by the length of the
polymethylene moiety between the two ammonium groups of
hydroxyl-containing alkylammonium gemini surfactants. In this
case, substrate hydrolysis was enhanced in the presence of addi-
tives with 10 and 12 methylene groups, while gemini surfactants
with shorter spacers inhibited the enzyme. Substrate concentra-
tion in the aqueous phase decreased with increasing the length of
1,8-Bis(tributylammonium)octane dibromide (bisBOAB): mp
ꢀ
¼ 110–112 C
1H-NMR (CD3OD, 200 MHz) d ¼ 0.92 (tr, 18H, 6CH3); 1.22–1.42
(m, 20H, 10CH2); 1.49–1.65 (m 16H, 8CH2); 3.11–3.23 (m, 16H,
8CH2).
1,4-Bis(tributylammonium)xylene dibromide (bisBAB): mp >
ꢀ
220 C
1H-NMR (CD3OD, 200 MHz) d ¼ 0.92 (tr, 18H, 6CH3); 1.22–1.40
(m, 12H, 16CH2); 1.49–1.65 (m 12H, 6CH2); 3.50–3.60 (m, 12H,
6N–CH2); 5.20 (s, 4H, 2Ar–CH2–N); 7.80 (s, 4H, Ar).
a-Chymotrypsin activity assay
the spacer and it was, again, the main factor leading to a reduc- The a-CT activity measurements have been carried out spec-
tion in the enzyme activity.
trophotometrically at 25.0 ꢁ 0.1 ꢀC, following the increase in
The present study aimed to gain a more extended insight into absorbance at 410 nm of the hydrolysis product of GPNA, p-
enzyme–cationic additive interactions. a-CT catalyzed hydrolysis nitroaniline (pNA). The molar absorption coefficient (3410) is
of GPNA was studied in the presence of three additives bearing 8800 Mꢂ1 cmꢂ1 in pure buffer and any variations in presence of
a
benzyl moiety on the ammonium ion. Benzyltributyla- the additives at the concentrations used in kinetic measure-
mmonium bromide (BzTBABr), benzyltrimethylammonium ments are evaluated and considered in rate values estimations.
bromide (BzTMABr) and the surfactant benzyldodecyldimethy- Measurements are performed with a Shimadzu UV-160A UV-VIS
lammonium bromide (BzDDABr) were selected. Moving forward spectrophotometer equipped with a thermostated cell.
on the studies of specic synthetic molecules for specic roles,
a-CT activity assay mixture was prepared in 0.1 M Tris–HCl
synthetic 1,8-bis(tributylammonium)octane dibromide (bisBOAB) buffer at pH 7.75; the enzyme concentration was always 0.2 mg
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mlꢂ1 (8 mM) and the concentration of the substrate GPNA was
2.5 ꢃ 10ꢂ3 M (unless otherwise specied). The reaction starts by
enzyme addition from stock solution (10 mg mlꢂ1) to a pre-
thermostated solution of substrate. The linear increase of
absorbance at 410 nm due to pNA formation is then recorded as
a function of time for 300 seconds; reaction rate of a-CT,
dened as moles of pNA formed per unit of time, is calculated
from the slope of the initial linear curve of pNA concentration
vs. time.
Results and discussions
Effect of cationic additives on a-chymotrypsin catalytic
properties
As previously reported,20 tetralkylammonium additives, with or
without a hydrocarbon chain, increased enzyme activity when
alkyl groups were rather bulky and hydrophobic. Inserting an
aromatic ring on additive head group could lead to a further
increase in hydrophobic/hydrophilic balance on additive
structure, so we performed kinetic studies on aromatic-derived
additives.
Benzyl-derived additives. The effect of BzTMABr, BzTBABr,
BzDDABr on a-CT catalytic properties was investigated and
results were compared with those previously obtained with
TMABr, TBABr and CTABr, respectively.15,20 BzDDABr, a surfac-
tant with a C-12 alkyl chain, was selected instead of the C-16
derivative because of the very low solubility of the latter.
Fig. 2 shows proles of a-CT activity as a function of additive
concentration, in the presence of BzTBABr, BzTMABr and
BzDDABr. In this plot, as in the following ones, the enzyme
activity is quoted as the ratio between the reaction rate in the
additive solution and in pure buffer.
In all cases, the introduction of a benzyl group into the
additive structure produced a superactivating effect on the
enzyme, represented by a bell-shaped curve as the additive
concentration increases. Regarding BzTBABr, two different
positive effects on enzyme activity were observed if compared
with the “homologue” TBABr. First, a higher superactivation
was attained since 12-fold increase in the reaction rate was
registered, compared to the 8-fold one previously reported with
TBABr (with respect to values in pure buffer). Moreover, the
maximum of activation was reached at a lower concentration
(0.15 M for BzTBABr vs. 0.4 M for TBABr). On the other hand,
a complete change in activity trend was observed in the pres-
ence of BzTMABr, if compared with its tetramethyl homologous
TMABr. TMABr produced only a slight and progressive increase
Kinetic parameters kcat and KM in pure buffer and in pres-
ence of additives were obtained from the linear regression
analysis of the double reciprocal Lineweaver–Burk plots in
a range of substrate concentration between 0.1 ꢃ 10ꢂ3 M and
2.5 ꢃ 10ꢂ3 M.
All sets of experiments were reproduced at least three times
and the differences between duplicates in each experiment were
always below 5%.
a-Chymotrypsin stability assay
The 0.2 mg mlꢂ1 enzyme solutions were prepared either in pure
buffer or in the presence of the additives and incubated in a water
bath at 25.0 ꢀC. Equal aliquots were periodically withdrawn and
injected to a thermostated substrate solution; the a-CT residual
activity was measured as described in the former section.
Molecular modelling studies
The possible binding poses of the tested additives in the
surrounding of the a-CT catalytic site were explored using the
FLAP (Fingerprints for Ligands and Proteins) soware, which is
developed and licensed by Molecular Discovery Ltd. (http://
www.moldiscovery.com). The docking procedure based on the
FLAP algorithm28 was rst used to generate the GRID Molecular
Interaction Fields (MIFs) for the a-CT cavity (pdb code: 4CHA,
chains A, B and C). The MIFs inspection allows to visualize and
quantify the hydrophobic and hydrophilic regions as well as the
regions where H-bond donors and acceptors might occur. In
this study, a FLAP pocket centered on the Ser-195 (erosion ¼ 15,
thickness ¼ 5) was dened to select the region for MIFs calcu-
lation and docking runs. The probes used to generate the
molecular interaction elds were H (shape), DRY (hydrophobic
interactions), N1 (H-bond donor) and O (H-bond acceptor)
interactions. Thus, a-CT cavity was used as a template, and the
binding poses of the additives were generated using FLAP in the
structure-based mode.29–31 A maximum of 50 different confor-
mations for each additive was generated and docked, to mimic
exibility. The obtained FLAP binding poses were ranked by the
Glob-Prod descriptor, accounting for the best compromise
between hydrophobic, hydrophilic, H-bond donor and H-bond
acceptor interactions, and the binding pose with the highest
similarity score was selected for each compound. In all cases,
Glob-Prod values corresponding to the selected poses was
greater than 0.9 (Glob-Prod scale from 0 to 1). In addition, the
10 top-ranked poses for each additive were visually inspected, to
evaluate the variability in result. Variability in the orientation of
the additives among the 10 top-ranked poses, when occurred, is
discussed.
Fig. 2 Effect of BzTBABr (:), BzTMABr (C) and, in the inset, BzDDABr
(-) concentration on the activity of a-chymotrypsin in 0.1 M Tris–HCl
buffer, pH 7.75 at 25.0 ꢀC.
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Table 1 a-Chymotrypsin kinetic parameters in the presence of addi-
tives and in pure buffer at 25.0 ꢀC
kcat(add)/kcat(b) was about 4-fold higher with respect to the value
in pure buffer, in a way similar to what we observed in our study
for the hydrolysis of GPNA.
Concentration,
M
KM,
mM
102 kcat
,
kcat(add)
kcat(b)
/
kcat/KM,
sꢂ1
Mꢂ1 sꢂ1
Enzyme kinetic parameters in the presence of BzTBABr have
been compared to those previously quoted with TBABr.20 The
increase in kcat obtained with both additives was the same, i.e.
0.31 sꢂ1 (20-fold with respect to pure buffer), but the super-
activation effect produced by BzTBABr was higher than TBABr,
i.e. 12.4 and 8-fold respectively. Such evidence could be
explained with the corresponding KM values, being the enzyme–
substrate affinity threefold lower in the presence of 0.4 M TBABr
(KM ¼ 6.1 mM) with respect to the benzyl-derived additive. As
already pointed out for DDBzABr, the observed effect could be
simply related to the lower concentration of BzTBABr which
produced the highest a-CT activation, considering that the
benzyl-derived additive could establish p–p interactions with
aromatic residues in enzyme catalytic site, promoting therefore
the association between the enzyme and the additive itself.
Diammonium-derived additives. As already pointed out,
hydrophobic/hydrophilic balance of the additive structure plays
a key role in determining the superactivation effect on a-CT.
Therefore, in view of a further improvement in the catalytic
performances of the enzyme, two new diammonium salts have
been properly designed and synthesized: 1,8-bis(tributyla-
mmonium)octane dibromide (bisBOAB) and 1,4-bis(tributyla-
mmonium)xylene dibromide (bisBAB). They both present two
tributylammonium head groups constrained by a different
spacer. BisBOAB possesses a saturated eight carbon atoms
chain separating the two ammonium groups, while in bisBAB
the same number of carbon atoms are arranged in a benzene
ring and so the hydrophobicity of the whole structure turn out
to be further increased. The proles of a-CT activity in the
presence of different concentrations of bisBOAB and bisBAB are
reported in Fig. 3.
Additive
—
—
0.44
1.37
1.99
0.49
1.46
8.70
31.10
6.70
—
33.2
63.5
156.3
136.7
BzTMABr
BzTBABr
BzDDABr
0.40 M
0.15 M
0.01 M
6.0
21.3
4.6
in a-CT activity, likely due to a simple salt effect,20 while a 5-fold
enzyme activation was gained with BzTMABr at a concentration
of 0.4 M.
Similar positive outcomes were obtained also in the presence
of a surfactant with an aromatic ring in the head group.
BzDDAB, unlike CTABr, not only produced a 3-folds a-CT
superactivation, but this effect was registered at a lower
concentration with respect to tetralkylammonium salts, i.e. 0.01
M, probably because the presence of a long hydrocarbon chain
could promote enzyme–additive association.
Kinetic parameters were also determined. Table 1 reports
enzyme–substrate affinity (Michaelis constant, KM), turnover
number (kcat), ratio between kcat values in the presence of
additive and in pure buffer (kcat(add)/kcat(b)) and kcat/KM values.
Experiments were performed at the additive concentration that
produced the maximum of superactivation.
In the presence of all investigated additives, enzyme–
substrate affinity decreased but at the same time higher kcat
values than pure buffer were observed. In previous studies,
similar changes in kinetic parameters are also reported for
TBABr aqueous solutions.20 To explain the positive effects re-
ported, it was supposed that the closeness of the additive to the
active site led to an increase in the hydrophobicity of the latter
and so to a higher nucleophilicity of the hydroxyl group of Ser-
195, mostly responsible for enzyme activity. On the other hand,
the registered decrease in the affinity between enzyme and
substrate could be explained either with direct interactions
between additive and substrate or with some negative effect of
the additive on enzyme conformation.
Both diammonium salts caused a marked increase in the rate
of the hydrolysis reaction with a 9-fold and a 6-fold super-
activation in the presence of bisBOAB and bisBAB respectively. In
Experimental data reported in Fig. 2 show a 12-fold activa-
tion in the presence of BzTBABr, while, as evident in Table 1, the
reaction rate ratio calculated from Lineweaver–Burk plot was
signicantly higher, i.e. about 20. Such disagreement is only
apparent, since kcat(add)/kcat(b) value is referred to saturation
conditions of substrate, which means too high concentrations
to be employed in the kinetic experiments. On the contrary, the
calculated reaction rate ratios in the presence of BzTMABr were
not notably different from the experimental ones, essentially
since the increase in KM value in this case was smaller. The
reported superactivation with the benzyl-derived surfactant,
BzDDABr, can be addressed to an increase in kcat, since the
enzyme–substrate affinity remained unchanged, probably
because of the very low additive concentration at which the
maximum of enzyme activation was gained. The same positive
effect of BzDDABr has been reported in literature for the
hydrolysis of p-nitrophenyl acetate,17 where the obtained
Fig. 3 Effect of bisBOAB (-) and bisBAB (C) concentration on the
activity of a-chymotrypsin in 0.1 M Tris–HCl buffer, pH 7.75 at 25.0 ꢀC.
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particular, a bell-shaped trend of activity was obtained by varying
bisBOAB concentration; the maximum superactivation value
reached was the same of the one in the presence of TBABr.
Nevertheless, as it could be expected, the concentration at which
the maximum activity was achieved was four times lower, prob-
ably because of the synergic effect of two tributylammonium
groups on the same single additive molecule.
As regards bisBAB, its poor solubility did not allow to obtain
data at concentrations higher than 0.2 M; but in any case the
rate of reaction achieved was lower than in the presence of
bisBOAB.
Based on the above reported results with benzylic additives, it
would be reasonably to expect a higher superactivating effect of
bisBAB on enzyme with respect to bisBOAB. The obtained oppo-
site result could be explained with the restricted conformational
freedom degree of the two bulky, hydrophobic head groups
separated by the aromatic ring, which could not allow them to
fully display their effect on instantaneous enzyme activity.
Kinetic parameters are also measured and the obtained
results are reported in Table 2.
Fig. 4 Residual activity of a-chymotrypsin in 0.15 M BzTBABr (C), 0.4
M BzTMABr (:), 0.01 M DDBzABr (;) and pure buffer (-) as a function
of storage time at 25.0 ꢀC. In the inset, the enzyme residual activity in
the first 24 h is also reported.
As already described in the case of monoammonium salts,
quite analogues effects on the enzyme catalytic properties were
produced also by the new diammonium additives, i.e. an increase
in the conversion rate of the substrate and, at the same time,
a reduction in the enzyme–substrate affinity. Again, a noteworthy
undervaluation in the superactivation effect, about 50–70%, was
observed with respect to the experimental data reported in Fig. 3,
and the same above accounted rationale could be provided to
explain such evidence, that is the lack of saturation conditions of
the substrate in the kinetic experiments.
Moreover, an interesting comparison could be stated
between the obtained data in 0.4 M TBABr and in 0.1 M bis-
BOAB. In both cases, enzyme relative activity was about 9-fold
higher than in pure buffer, but the presence of monoammonic
additive TBABr in the solution would have led to an instanta-
neous activity of a-CT signicantly higher than in bisBOAB, if
Enzyme stability
Enzyme stabilization is one of the most important topics in
basic and applied enzymology and understanding any rela-
tionship between the structure of the additive and its effect on
enzyme stability is of particular relevance.
Benzyl-derived additives. Fig. 4 depicts the time course of a-
CT activity in pure buffer and in aqueous solutions containing
BzTBABr, BzTMABr and DDBzABr as well. The ratio between the
instantaneous specic reaction rate and the one in pure buffer
at zero time is reported as function of storage time in order to
easily compare the deactivation kinetics in the different media
and to display superactivity preservation during storage time. In
the inset, the ratio of instantaneous to initial rate in the rst 24
ꢀ
h is also reported. Stability was monitored at 25.0 C and the
investigated additive concentrations were those reported in the
caption of the gure.
saturation conditions could be achieved, since their kcat(add)
/
kcat(b) ratios were 21 and 13, respectively.
In the absence of additives and in the rst 24 h, a-CT lost 25%
of its initial activity; then, activity continued to decrease, until,
aer 30 days, the enzyme retained less than 30% of its activity.
Enzyme deactivation in the presence of the surfactant
DDBzABr was even faster, since relative residual activity
decreased of about 30% in the rst 8 h, and, aer 96 h, it was
even negligible. On the contrary, the benzyl-derived salts allowed
the enzyme to retain almost all of its activity during the rst
hours of incubation, and the superactivation levels observed in
the kinetic experiments were maintained. Aer one month of
incubation, the percentage of residual enzymatic activity was
lower in the presence of BzTBABr and BzTMABr than in pure
buffer, between 15 and 20%. Therefore, although the presence of
a benzyl group as substituent on additive head group at rst
produced an increase in instantaneous activity, in the long period
it speeded up the a-CT deactivation. Nevertheless, at least in
BzTBABr solutions, aer 30 days residual activity was about 3
times higher than that in pure buffer at time zero, which means
that the enzyme was still superactivated.
As already pointed out, the degree of conformational
freedom of the additive structure certainly plays a relevant role
in the positive effect of the additive itself on enzyme activity.
Therefore, notwithstanding the notable lower concentration at
which the maximum of superactivation was reached, it can be
hypothesized that the just described effect was dependent on
the restricted mobility of the two tributylammonium head
groups in bisBOAB, if compared with the single, totally free
tetrabutylammonium ions.
Table
2 a-Chymotrypsin kinetic parameters in the presence of
additives and in pure buffer at 25.0 ꢀ
C
Concentration,
M
KM,
mM
102 kcat
sꢂ1
,
kcat(add)
kcat(buffer)
/
kcat/KM,
Additive
Mꢂ1 sꢂ1
—
—
0.1 M
0.1 M
0.44
1.49
1.86
1.46
12.0
19.3
—
8.2
13.2
33.2
80.5
103.8
BisBAB
BisBOAB
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additives. In particular, it was hypothesized that additives could
interact with the enzyme making the region close to the catalytic
triad more hydrophobic, and thus favoring the catalytic activity.
Therefore, we attempted to rationalize this hypothesis in
silico, studying the possible binding modes of the tested addi-
tives in the catalytic site environment. Based on the experi-
mental conditions used for our tests, in which the additives
were added to the enzyme prior to substrate addition, we
decided to model the interaction between each additive and the
enzyme in the absence of the substrate. The X-ray structure of
un-complexed a-CT (PDB code: 4CHA) was selected because of
˚
its high quality resolution (1.68 A) and because already used as
a valuable structure for modelling studies.32–35 The FLAP
(Fingerprints for Ligands and Proteins) algorithm has been
extensively described elsewhere,28 and was used rst to identify
the cavity surrounding the catalytic triad, and then to evaluate
the most favorable binding poses of the studied additive in the
Fig. 5 Residual activity of a-chymotrypsin in 0.1 M bisBOAB (C), 0.1 M
bisBAB (:) and pure buffer (-) as a function of storage time at 25.0 ꢀC. selected region.
In the inset, the enzyme residual activity in the first 24 h is also
Briey, FLAP is a virtual-screening and model-development
reported.
method based on 3D molecular similarity, measured through
common Molecular Interaction Field (MIF) volumes.28–31 In
a structure-based approach, MIFs representing the regions of
protein cavity (here a-CT) suitable for hydrophobic, hydrophilic,
H-bond donor or H-bond acceptor interactions are compared
with analogue MIFs generated on the ligand structure (here the
additive molecule); thus, the similarity between MIFs of the
protein and MIFs of the ligand is expressed in terms of a simi-
larity score (here the Glob-Prod score). Since several binding
poses might be produced by FLAP for a ligand, in this study for
each ligand the correspondent FLAP results were ranked by the
Glob-Prod similarity score, and the pose with the highest
similarity score was extracted.
Benzyl-derived additives. Fig. 6A reports the hydrophobic
regions in terms of MIF (green volumes) close to the catalytic
moiety of the enzyme in the un-complexed state, while the FLAP
binding poses for benzyl-derived additives are shown in Fig. 6B–D.
When hydrophobic MIFs are generated in FLAP, it is possible
to dene which amino acids contribute to a local hydrophobic
effect. Concerning the a-CT cavity, in addition to a minor
hydrophobic region generated by the aromatic ring of H57, the
main hydrophobic region is generated by W215, directly facing
the catalytic region. A further additional elongated hydrophobic
region was observed closely (Fig. 6A). The modelling studies for
the benzyl-derived additives showed that BzTBABr, BzTMABr
and BzDDABr favorably orient their aromatic moiety towards
W215, leading to an efficient p–p stacking. The orientation of
the benzyl-derived additives is then inuenced by the other
surrounding hydrophobic regions, depending on the nature of
the additive. For example, for BzTMABr several binding poses
with very similar FLAP similarity score (and thus to be consid-
ered almost equivalent) were obtained, preserving the interac-
tion with W215 but having the trimethylammonium group
variably oriented (data not shown). Fig. 6B illustrates the top-
ranked pose for BzTMABr highlighting that the trimethy-
Diammonium-derived additives. Finally, a-CT was incubated
in the presence of 0.1 M bisBOAB and 0.1 M bisBAB and the
enzyme residual activity was monitored as a function of time.
The corresponding proles are showed in Fig. 5 and data in
pure buffer are also reported for comparison. As in Fig. 4, the
inset shows the ratio of instantaneous to initial specic reaction
rate in the rst 24 h.
In the presence of both the additives, only a slight loss of
enzymatic activity was observed in the rst 48 hours, 10 and 20%
for bisBOAB and bisBAB respectively, compared with 30%
decrease in pure buffer. Interestingly, in the long period, trends
of residual activity in the presence of bisBOAB and the homo-
logue monoammonic TBABr are quite comparable,20 since in the
presence of bisBOAB enzyme retained almost 45% of its initial
instantaneous activity even aer one month of incubation.
As regards bisBAB, aer the rst 48 h the deactivation rate
was markedly higher than those in pure buffer and in bisBOAB
and, aer 30 days, the residual activity was only about 16% of
the initial value. However, although the superactivation effect
was almost completely lost in bisBAB, aer one month the
enzyme activity still remained higher than the starting value in
pure buffer.
The obtained results with the two di-tributylammonium salts
suggested that, even though the degree of exibility of the spacer
did not strongly affect enzyme superactivation, it seems to be of
primary importance in determining its deactivation rate. More-
over, the aromatic ring itself could negatively affect a-CT stability,
as suggested by the corresponding experiments with the mono-
ammonic benzyl-derived additives (see Fig. 4).
Molecular modelling studies
Experimental data collected suggested that the enzyme super- lammonium group, due to its small size and its low hydro-
activation effect could be related to a possible variation of the phobicity, cannot favorably interact with other hydrophobic
catalytic cavity environment upon interaction with certain regions. On the contrary, the best binding pose (highest
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Fig. 6 Modelling of the a-CT/additive interaction using FLAP. (A)
Representation of the hydrophobic MIF (green volumes) close to the
a-CT catalytic site. (B–D) FLAP binding poses for BzTMABr, BzTBABr
and BzDDABr, respectively.
Fig. 7 FLAP binding poses for TBABr (A), CTBABr (B), bisBOAB (C) and
bisBAB (D).
Diammonium-derived additives. Modelling studies were
extended to the two novel dicationic salts, bisBOAB and bisBAB,
and also to TBABr and CTBABr for comparative purpose. The
top-ranked binding poses by Glob-Prod similarity score for the
four compounds are reported in Fig. 7.
According to our model, notwithstanding the lack of an
aromatic moiety, the tributylammonium groups of the additives
are positioned in the hydrophobic MIF of W215. Of course, the
extent of the additive/W215 chemical interaction results to be
weakened. However, this W215 anchor point still allows the
additive to be located close to the hydrophobic region of H57,
producing an increase in the hydrophobicity of the active site
and, consequently, enzyme superactivation. In case of small-
size hydrophobic additives, such as TBABr and BzTBABr, their
rigidity force them to be similarly located close to the catalytic
similarity score) for BzTBABr is reported in Fig. 6C, with the
shown orientation being preserved among the 10 top-ranked
FLAP poses. In this case, the tributylammonium moiety
perfectly points towards the hydrophobic region generated by
H57, due to its more hydrophobic nature and to the rigid
structure of this additive. Finally, BzDDABr displayed a similar
binding pose with respect to BzTBABr (Fig. 6D); however, the
high exibility of the alkyl chain allows the latter to be oriented
either towards the H57 hydrophobic MIF (Fig. 6D) or towards
the elongated one far from the catalytic triad (data now shown).
A comparison of the FLAP binding poses for the three benzyl-
derived additives suggests that all the benzyl-derived additives
increase the hydrophobicity of the active site, but at different
level. In particular, BzTBABr is the compound able to most effi-
ciently increase the overall hydrophobicity of the a-CT catalytic
site. Indeed, it possesses three chemical features that force the
compound to be oriented towards the catalytic site: (1) the
aromatic portion to maximize the interaction with W215; (2) the
tributylammonium group at a distance from the aromatic moiety
that perfectly matches the hydrophobic region by H57; (3) a rigid
nature that stabilizes this hypothetical binding mode. As a main
result, the orientation of the tributylammonium group towards
the catalytic triad might increase the overall hydrophobicity of
that region, without hampering the substrate binding.
triad region, and accordingly their kcat are very similar (kcat
31.0 ꢃ 10ꢂ2 sꢂ1 and 31.1 ꢃ 10ꢂ2 sꢂ1, respectively).
¼
Concerning bisBOAB and TBABr, as previously reported the
enzyme superactivation in solutions upon their addition was very
similar, but the concentration at which the maximum is achieved
in the presence of the dicationic salt was four times lower than
TBABr. Modelling studies point out two effects that could explain
the experimental behavior. Firstly, the symmetric nature of bis-
BOAB doubles the chances to get a favorable binding with the
enzyme. In addition, the more hydrophobic bisBOAB can
strongly interact with the hydrophobic regions in the protein
cavity, stabilizing the orientation tributylammonium group
toward W215. Consequently, a lower number of additive mole-
cules is needed to produce the same superactivating effect.
However, the hydrophobic small-size features of TBABr seemed
to more efficiently increase the hydrophobicity of the catalytic
site with consequent increase of the kcat (31.0 ꢃ 10ꢂ2 sꢂ1 for
This analysis of the in silico models in Fig. 6 nicely correlates
with the experimental kinetic parameters. In fact, as reported in
Table 1, kcat in the presence of BzTMABr and BzDDABr is about
5–6-fold higher than that in pure buffer, while in BzTBABr
solutions the more hydrophobic environment could be
responsible for the increase of kcat of about 20-fold.
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TBABr and 19.3 ꢃ 10ꢂ2 sꢂ1 for bisBOAB). A plausible hypothesis,
Results suggested that enzyme catalytic properties could be
as previously discussed for BzDDABr, is that the high exibility of strongly affected not only by the charge, the size and the
BisBOAB can be responsible for the lower hydrophobic effect at hydrophobic/hydrophilic balance of additive head group, but
the catalytic triad region. Indeed, the long and exible spacer also by specic interactions between enzyme and denite
between the two tributylammonium moieties can interact with groups within the additive structure.
either the hydrophobic region generated by H57 or the more
Molecular modelling studies performed suggest that W215
extended one diagonally located (Fig. 6A), and according to the could be a key residue to anchor additives close to the catalytic
FLAP poses the second one seems to be more favorable (the pose site. According to the in silico evaluations, the a-CT super-
with highest similarity score is diagonally oriented). Although in activation by small size additives with reduced exibility could
a dynamic context the spacer could switch between the two be explained by the optimal exposition of the tributyl group
locations, the overall effect is a lower hydrophobic contribution towards the catalytic site, once the molecule is anchored to
on the catalytic triad.
W215. This binding mode seems to be responsible for an
In agreement with this hypothesis, CTBABr displayed a kcat increased hydrophobicity of the catalytic site, resulting in an
value very similar to that of bisBOAB and (kcat ¼ 19.5 ꢃ 10ꢂ2 sꢂ1 increased kcat value. Contrarily, more exible additives and
and 19.3 ꢃ 10ꢂ2 sꢂ1, respectively). Indeed, as for BzDDABr, the large rigid ones displayed a lower superactivation effect due to
high exibility of the alkyl chains of CTBABr and bisBOAB alternative binding modes for the alkyl chains.
actually allows a number of similarly ranked binding poses,
with the aliphatic chains being oriented towards the H57
hydrophobic MIF or towards the elongated one far from the
catalytic triad. Thus, Fig. 7B and C are representative of binding
Acknowledgements
`
The authors thank the Ministero per l'Universita e la Ricerca
mode with the highest similarity score. However, the visual
inspection of the ten top-ranked poses for these compounds
proved that in all of them a tributylammonium moiety is
located in correspondence of W215. Thus, an extensive exi-
bility of the additives generated by long alkyl chains seems to
induce an unfavorable effect on the kcat, both in the benzyl-
derived and in the diammonium-derived series.
Although exibility appears to be detrimental for kcat, the
more rigid bisBAB also displayed a low effect, with a kcat value of
12.0 ꢃ 10ꢂ2 sꢂ1. Fig. 7D illustrates the FLAP binding pose for
this compound. It is noteworthy that bisBAB, although a more
rigid compound, displays the worst interaction with W215,
probably due to its large size that make its direct exposition
towards the catalytic site more difficult.
Scientica e Tecnologica, MIUR (Rome, Italy) [PRIN “Pro-
grammi di Ricerca di Interesse Nazionale” 2010–2011, no.
2010FM738P] and Regione Umbria (POR FSE 2007–2013,
Risorse CIPE, Perugia, Italy) for funding.
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