Insight into the esterase like activity demonstrated by an imidazole appended self-assembling hydrogelator

Article abstract of DOI:10.1039/c5cc04281j

A low molecular weight hydrogelator with a covalently appended imidazole moiety is reported. Capable of percolating water in the pH range of 6 to 8, it proves to be an efficient catalyst upon self-assembly, showing Michaelis-Menten type kinetics. Activities at different pH values correlated with dramatic structural changes were observed. It can hydrolyse p-nitrophenyl acetate (pNPA) as well as inactivated esters, and l and d-phenylalanine methyl esters. The enhanced activity can be related to the conglomeration of catalytic groups upon aggregation resulting in their close proximity and the formation of hydrophobic pockets.

Full text of DOI:10.1039/c5cc04281j

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DOI: 10.1039/C5CC04281J
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Insight into the esterase like activity demonstrated by
an imidazole appended self-assembling hydrogelator.
Cite this: DOI: 10.1039/x0xx00000x
Nishant Singh,a Maria P. Conte,b R. V. Ulijn,b,c* Juan F. Miraveta* and Beatriu
Escudera*
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
nanostructure and multivalency on the catalytic performance.3 In this
A
low molecular weight hydrogelator with covalently
communication we report low molecular weight hydrogelator (1)
with covalently linked imidazole moieties demonstrating esterase
like activity. The ordered arrangement of the catalytic centres upon
selfꢀassembly of molecules into a fibrillar network is taken
advantage of to attain improved catalytic activity when compared to
the nonꢀaggregating analogue. In contrast to previously reported
examples, 1 is a small synthetically simple compound that is able to
hydrolyse not only activated but also nonꢀactivated aminoacyl
esters.3
appended imidazole moiety is reported. Capable of
percolating water in the pH range of 6 to 8, it proves to be an
efficient catalyst upon self-assembly, showing Michaelis-
Menten type kinetics. Activities at different pH correlated to
dramatic structural changes observed. It can hydrolyse
nitrophenyl acetate ( NPA) as well as inactivated esters,
and -phenylalanine methyl esters. The enhanced activity
p-
p
L
D
can be related to the conglomeration of catalytic groups upon
aggregation resulting in their close proximity and the
formation of hydrophobic pockets.
Mimicking and emulating enzymes by means of synthetic catalysts
is currently an area of huge interest.1Tertiary structure allows various
functional groups to be precisely arranged in threeꢀdimensional
space, creating sites to bind substrates and positioning chemical
functional groups to enable catalysis. Prediction and synthesis of
enzyme performance from de novo designed primary sequence able
to fold and function accordingly is currently not possible. However it
is possible to attain enzyme like activity by using smaller selfꢀ
assembling molecules or peptides owing to the ordered arrangement
of the catalytically active sites/functional groups. Several groups
have demonstrated esteraseꢀlike activity using metal based
nanoparticles, micelles or dendrimers with large molecular weights
and mostly with spherical symmetry.2 Histidineꢀcontaining peptide
nanofibrillar systems have been reported recently as catalysts for
activated ester hydrolysis (2,4ꢀdinitrophenyl acetate and pꢀ
nitrophenyl acetate) and have revealed the role of the fibrillar
Scheme 1: Selfꢀaggregating compound 1and nonꢀaggregating analogue 2.
Compound 1 with a minimum gel concentration of 5.7 mM was
tested for the esterase like activity using pNPA as substrate. pNPA
upon hydrolysis yields chromogenic pꢀnitrophenolate anion which
can be monitored at 400nm using UV spectroscopy (see ESI for
details). The initial parts of the kinetic curves for hydrolysis at
different concentrations of substrate were fitted to MichaelisꢀMenten
equation for respective pH values. The obtained KM, kcat and Vmax
values are presented in Table 1.
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Table 1: Obtained KM, kcat and Vmax values of hydrolysis of pNPA by1.
pH values 6 and 6.5, cannot simply be attributed to the contribution
of the fraction of deprotonated imidazole. Instead, cooperativity
between both protonated and deprotonated imidazole groups is likely
responsible.
pH 6
pH 6.5
pH 7
pH 7.5
pH 8
kcat(sꢀ1
KM(mM)
Vmax(ꢁmolsꢀ1
)
×102
2.6±0.4
3.3±1.1
8.8
2.5±0.6
3.1±1.6
8.6
2.1±0.4
4.0±1.5
7.3
2.4±0.7
3.9±1.6
8.1
2.5±0.6
3.7±1.3
8.3
)
The solubility (Ksol) of compound 1 was obtained by NMR
experiments for pH 6, pH 7 and pH 8 are 2.85 mM, 1.95 mM and
0.95 mM respectively (Fig. S4). The Ksec (second order rate
constant) values observed at these pH respective non aggregating
concentrations of compound 1, were significantly lower than at the
selfꢀassembling concentration (Fig. S3). Thus, highlighting the fact
that observed catalysis is not by the molecules in solution but due to
the catalytically active fibers. This was further confirmed by the
catalytic activity observed for the nonꢀassembling analogue 2. The
Fig. 1: (a) UV kinetic profile of pNPA (1.4 mM) hydrolysis at 400 nm
catalysed by 0.17 Mm of compound 1 gel, compound 2 and background
hydrolysis at pH 6.5. (b) kcat/KM values obtained by compound 1 at different
pH values (black bars) and Ksec values for 2 (white bars, * not determined).
K
sec for 2 at pH 6.5, pH 7 and pH 8 are 0.5 Mꢀ1sꢀ1, 0.7 Mꢀ1sꢀ1 and 2.2
Mꢀ1sꢀ1 which are significantly lower than for 1 under identical
conditions The obtained linear relationship between velocity of
product formation and substrate concentration reflected lack of any
binding events unlike 1 (Fig. 1a and S2b).
The calculated pKa values for aggregated compound 1 are 5.9 and
6.6, with monoprotonated species in abundance in this range (see
Fig. S1).This drop in pKa value to 5.9 is only observed for the
aggregated form of 1, as in solution the pKa values are similar (6.2
and 6.3). This indicates the increase in basicity of one of the two
imidazole moieties in molecule 1 only in the selfꢀassembled state.
The catalytic efficiency (kcat/KM) for imidazole molecules in solution
is known to drop significantly at lower pH values. 2a,3,4a The higher
amount of protonated imidazole moieties are held responsible for
this which normally are not catalytically active. To our surprise for
the pH 6 and 6.5 we don´t observe this drop, infact a slight rise in the
catalytic efficiency when compared to higher pH values like 7 and
7.5. This unusual catalytic efficiency around the pka values of selfꢀ
assembling 1 suggests the involvement of both protonated and
deprotonated imidazoles in the catalysis, often referred to as a
cooperative mechanism in the prior art (Fig. S2c and d).2a,3,4a
Fig.4 (a) Product formation upon hydrolysis of Lꢀphenylanine methyl ester
(12.3mM) by 1 (5.6mM) in the span of 80 hours as observed by HPLC. (b)
MichaelisꢀMenten fitting at different concentrations for pH 7.
Well above the pKa only the deprotonated imidazole residues are
present and held responsible for the hydrolysis. The catalytic
efficiency at pH 8 only due to the deprotonated imidazole groups is
7.37 Mꢀ1sꢀ1. We know from the potentiometric titration curve that the
monoꢀprotonated imidazole is present in fourꢀfold excess compared
to the deprotonated imidazole at pH 6.5 (Fig. S1). According to these
values, the extrapolated catalytic efficiency of the deprotonated
imidazole at pH 6.5, assuming that the protonated imidazole is not
active should be 1.65 Mꢀ1sꢀ1 whereas that observed is 8.09 Mꢀ1sꢀ1.
Hence, based on these observations the catalytic efficiency at lower
Owing to the fast hydrolysis trend shown by 1 we anticipated it to be
capable of hydrolysing even stronger non activated ester linkages
such as methyl esters. We chose L and Dꢀphenyalanine methyl esters
as our substrate and followed its hydrolysis by HPLC (see ESI for
details). As expected, these substrates did not undergo any
noticeable background hydrolysis at pH 7 whereas, with 33 mol% of
1 as catalyst, we could observe commendable hydrolysis rate of the
methyl esters (Fig. 4a and S8). The calculated catalytic efficiency for
the hydrolysis of L and Dꢀphenyalanine methyl esters by 1 are
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1.2×10ꢀ3 Mꢀ1hrꢀ1 and 1.7×10ꢀ3 Mꢀ1hrꢀ1 respectively. The Ksec (2.5×10ꢀ also evident from the circular dichroism (CD) contours (Fig. 3(a)
4
Mꢀ1hrꢀ1) for 2 is an order of magnitude smaller than for 1 and S9). While moving from pH 8 to pH 6 an increase in ellipticity
exemplifying the aggregation effect, even though there doesn´t seem together with an apparent redꢀshift is observed in the CD spectra.5a,b,d
to be any stereoselectivity shown by 1 (Fig. 4b). Thus, the hydrolysis
of nonꢀactivated ester at the physiological pH, which to our
knowledge has not been demonstrated before, reflects the fact that
aggregation can lead to new emergent catalytic abilities even in
small molecules owing to the high local concentration of the
catalytic sites.
Fig. 3: (a) CD spectra obtained for gels at different pH values and (b)
fluorescence emission of ANS in gels and background at different pH values.
These kinds of spectral shifts to longer wavelengths are reported for
Fig.2: TEM images of gels at (a) pH 6 (scale bar: 50nm) (b) pH 6.5 (scale
twisted fibers having πꢀπ* transitions with larger dipole moments
bar: 100nm (Inset: zoomed image of twisted fibre) (c) pH 7 (scale bar:
which is expected to be the case in the protonated imidazole more
than the deprotonated ones.5c This information obtained from CD
0.5µm) (d) pH 8 (scale bar: 0.5µm)
and IR are in agreement with the TEM images of the catalytic gels
which switch from twisted helical fibers (pH 6 and 6.5) to flat belt
like structures (pH 7 and 8).
A series of experiments were performed to obtain the structure
activity relationship. The nanoscale structures of the selfꢀassembled
networks of gels as observed by TEM differ depending on pH values
(Fig. 2). TEM images reveal helical fibres of 15ꢀ20 nm width at pH
6 and 6.5. The higher Vmax values for pNPA hydrolysis obtained at
these pH values can be attributed to the observed high aspect ratio
fibres, allowing higher extent of substrate binding thus releasing
more product. At pH 7 and 8 the molecules aggregate in flat beltꢀlike
structures which are 0.5µm wide and several micrometers long, with
lower aspect ratio. The variation in morphologies based on pH can
be attributed to the change in number of protonated imidazole
residues. The repulsion between higher number of protonated
imidazole present at pH 6 and 6.5 may result in stacking of
molecules with higher slip angle between them; this gives rise to the
twists observed in the nanofibers. This is not seen further away from
the pKa by increasing the pH due to the significantly lower number
of protonated imidazole residues.
In order to evaluate the presence of hydrophobic binding pockets, we
probed the gels at different pH values with 1ꢀanilinonaphthaleneꢀ8ꢀ
sulphonate(ANS), used extensively for this purpose in protein
structure studies.6 When ANS was introduced in hydrogel 1, an
apparent blue shift along with the rise in fluorescence was observed
confirming the presence of hydrophobic pockets (Fig. 3(b)). These
hydrophobic binding pockets could be formed by the central alkyl
chain as well as by the Lꢀvaline isopropyl residues in 1. The
molecular rigidity introduced by the two amide groups could be
balanced by the flexibility of the alkyl spacer as well as the
rotational freedom of the imidazole end fragments. Overall, a
dynamic binding pocket could be confirmed in the vicinity of the
catalytic fragment. It is also remarkable the effect of the Lꢀvaline
residue as compound 2 lacking this amino acid fragment does not
even aggregate under similar conditions. Moreover, the fluorescence
intensity at pH 6 and 6.5 is higher and more blue shifted (472 nm for
The amide I´ band in the 1630ꢀ1638 cmꢀ1 region of IR for different
pH values denote the presence of βꢀsheet like structures, which is
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DOI: 10.1039/C5CC04281J
pH 6 and 474 nm for pH 6.5) than for the rest of the pH values, after millions of years. In fact, it has been proposed that short selfꢀ
suggesting stronger binding of the ANS and supporting the lower KM assembled peptides could have played a relevant role in the early
values obtained for the pNPA hydrolysis at these pH values. In stages of the origin of life.7 In this context, selfꢀassembled catalytic
summary, the kcat observed for pNPA hydrolysis shows little peptides could have been potential precursors of enzymes.
variation with pH, with the change in catalytic efficiency probably
Acknowledgments
due to differences in substrate binding. Although the errors in KM are
This work was supported by the EU Marie Curie ITNꢀSmartnet and the
significant, a clear trend is observed. This behaviour can be
Ministry of Economy and Competitiveness of Spain (Grant CTQ2012ꢀ
attributed to the difference in availability of binding pockets for each
37735). N.S. and M.P.C. thank EU for an ESR Marie Curie contract.
morphology. It is remarkable that a simple low molecular weight
Notes and references
selfꢀassembling compound bearing a minimal peptidic fragment is
able to catalyse the hydrolysis of an activated ester as efficiently or
even better than the few previously reported examples of longer selfꢀ
assembled peptides.3 For instance, Stupp et al.3areported a rate
constant of 1.67 x 10ꢀ2 sꢀ1 and a KM of 0.4 mM for a bisꢀhistidine
aDepartament de Química Inorgànica i Orgànica, Universitat Jaume I,
12071 Castelló, Spain Fax: +34 964728214; Tel: +34 964729155; Eꢀ
mail: miravet@uji.es, escuder@uji.es.
bWestCHEM/Department of Pure & Applied Chemistry, University of
Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, U.K.
cAdvanced Science Research Center (ASRC) and Hunter College, City
University of New York, 85 St Nicholas Terrace, New York NY10027,
United States.
containing decapeptide and
a less efficient tetradecapeptide
†
Electronic Supplementary Information (ESI) available: experimental
details on synthesis and characterisation of compounds and catalytic
experiments. See DOI: 10.1039/c000000x/
containing one histidine residue was described by Liang et al.3c(kcat
0.19 x 10ꢀ2 sꢀ1 and KM 21.68 mM). The same group reported a
shorter analogue (FmocꢀFFHꢀCONH2) with lower reaction rate than
in the case of compound 1 (kcat 0.14 x 10ꢀ2 sꢀ1) but with a stronger
binding ability (KM 0.76 mM).3b Related results have been also
reported by Korendovych et al. for amyloidogenic heptapeptides
bearing H residues that after selfꢀassembly are able to coordinate
Zn+2 and efficiently catalyse the hydrolysis of pNPA.3d This example
however follows a different catalytic mechanism based on metal ion
Lewisꢀacid catalysis. All these examples highlight the undoubtable
link between selfꢀassembly and emergence of catalysis which is also
exemplified by compound 1.
References:
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In summary, we have reported an example of
a catalytic
supramolecular hydrogel in which binding and catalysis take place in
reaction sites selfꢀconstructed solely with nonꢀcovalent interactions.
We have been able to identify the probable structureꢀactivity
relationship between the different selfꢀassembled structures and the
subsequent catalytic performances of the gels. The results indicated
that the presence of hydrophobic pockets, higher aspect ratio of
fibres and the balance between the protonated and deprotonated
imidazole can be the key factors for improved hydrolysis efficiency
compared to related nonꢀaggregating catalysts. It is remarkable the
fact that enzymeꢀlike catalytic behaviour emerges after selfꢀ
assembly of small and simple molecular components. It is worth to
mention that very simple lowꢀmolecular weight compounds can
approach, although modestly by now, what evolution has achieved
Korendovych, Nature Chem.2014, 6, 303ꢀ309.
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