Modulation of the catalytic behavior of α-chymotrypsin at monolayer-protected nanoparticle surfaces

Article abstract of DOI:10.1021/ja064433z

Amino-acid-functionalized gold clusters modulate the catalytic behavior of α-chymotrypsin (ChT) toward cationic, neutral, and anionic substrates. Kinetic studies reveal that the substrate specificity (k_cat/K _M) of ChT-nanoparticle co

Full text of DOI:10.1021/ja064433z

Published on Web 10/25/2006
Modulation of the Catalytic Behavior of r-Chymotrypsin at
Monolayer-Protected Nanoparticle Surfaces
Chang-Cheng You, Sarit S. Agasti, Mrinmoy De, Michael J. Knapp, and
Vincent M. Rotello*
Contribution from the Department of Chemistry, UniVersity of Massachusetts, 710 North
Pleasant Street, Amherst, Massachusetts 01003
Received June 22, 2006; E-mail: rotello@chem.umass.edu
Abstract: Amino-acid-functionalized gold clusters modulate the catalytic behavior of R-chymotrypsin (ChT)
toward cationic, neutral, and anionic substrates. Kinetic studies reveal that the substrate specificity (kcat
/
KM) of ChT-nanoparticle complexes increases by ∼3-fold for the cationic substrate but decreases by 95%
for the anionic substrate as compared with that of free ChT, providing enhanced substrate selectivity.
Concurrently, the catalytic constants (kcat) of ChT show slight augmentation for the cationic substrate and
significant attenuation for the anionic substrate in the presence of amino-acid-functionalized nanoparticles.
The amino acid monolayer on the nanoparticle is proposed to control both the capture of substrate by the
active site and release of product through electrostatic interactions, leading to the observed substrate
specificities and catalytic constants.
Introduction
possessed by small molecule ligands: First, they may provide
large surface areas for efficient protein binding, as typically >6
nm2 of buried surface are involved in forming protein-protein
interfaces in nature.5 Second, multivalent functionalities can be
grafted on the materials to meet the structural complexity of
proteins. Among diverse nanoscale entities, monolayer-protected
nanoparticles are extremely attractive due to their ease of
synthesis and structural diversity and have found successful
applications in the interaction with proteins.6,7
Modulation of enzyme activity provides a potent means to
gain control over cellular processes such as signal transduction,
DNA replication, and metabolism. On the other hand, aberrant
activities of some enzymes can lead to the development of
numerous diseases and disorders including cancers and human
immunodeficiency virus (HIV) disease.1,2 From this point of
view, the modulation of enzyme activity is of great importance
in therapeutics and pharmaceutics.3 Synthetic small organic
molecules (e.g., most drugs) have been used to inhibit the
enzyme activity through the direct interaction with the active
sites of enzymes.4 Recent advances in nanoscale materials offer
a new pathway for regulating enzyme behavior through surface
interactions, providing a promising alternative to small molecule
inhibition. Nanomaterials possess at least two attributes not
As a representative serine protease, R-chymotrypsin (ChT)
provides an excellent enzyme model for studying the modulation
of activity due to its well-defined structure and extensively
characterized enzymatic properties.8 As illustrated in Figure 1a,
the active pocket of ChT is surrounded by a ring of positively
charged residues. Meanwhile, a number of hydrophobic “hot
spots” are distributed on the surface. Such structural features
allow the electrostatic interaction as well as hydrophobic
interaction with receptors possessing complementary surfaces.
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10.1021/ja064433z CCC: $33.50 © 2006 American Chemical Society

Nanoparticle Modulation of Chymotrypsin Behavior
A R T I C L E S
Figure 1. (a) Molecular structure of R-chymotrypsin. (b) Chemical structure of amino-acid-functionalized gold nanoparticles and SPNA-derived
substrates. (c) Schematic representation of monolayer-controlled diffusion of the substrate into and the product away from the active pocket of nanoparticle-
bound ChT.
Macrocyclic hosts9 and surfactant micelles10 have been used
so far to improve the activity and stability of ChT in organic
solvents through surface binding.
study, we systematically investigate the enzymatic kinetics of
ChT in the presence of various amino-acid-functionalized gold
nanoparticles (Figure 1b). The structural diversity of amino acids
allows us to probe the role of various functional groups in
controlling enzymatic function. With SPNA derivatives bearing
positive, neutral, or negative end groups as substrates, we have
demonstrated that the interaction between the nanoparticle
monolayer and the substrates affects both kcat and KM values,
resulting in an enhanced substrate selectivity for nanoparticle-
bound ChT. Such results are ascribed to the monolayer-
controlled uptake of the substrates and diffusion away of the
products through electrostatic interactions (Figure 1c). In nature,
electrostatic interactions play an important role in the uptake
of substrates by many diffusion-controlled enzymes including
superoxide dismutase (SOD),14 acetylcholinesterase (AChE),15
and triosephosphate isomerase (TIM).16 The surface recognition
of ChT by charged nanoparticles provides a means to mimic
this strategy by tuning enzyme-substrate association.
We have demonstrated that carboxylic-acid-functionalized
nanoparticles can effectively associate with R-chymotrypsin
through surface complementary charges to afford supramolecular
complexes, resulting in the inhibition of its enzymatic activity
toward anionic substrates such as N-succinyl-L-phenylalanine
p-nitroanilide (SPNA).11,12 This phenomenon has been attributed
to the obstruction of the active site by the complex formation
which reduces the accessibility of the substrates. Interestingly,
in a follow-up study13 we found that the association of ChT
with the simple carboxylic acid monolayer-protected nanopar-
ticles results in differential behavior with varying substrates.
Nanoparticle-bound ChT showed no activity change toward the
positively charged substrates, in contrast to the significant
activity depression toward anionic substrates. In the current
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Enzyme Microb. Technol. 2003, 32, 356-361. (c) Tremblay, M.; Cote, S.;
Voyer, N. Tetrahedron 2005, 61, 6824-6828.
Results and Discussion
Our previous study demonstrated that amino-acid-function-
alized nanoparticles and ChT can form high affinity complexes
in dilute sodium phosphate buffer (5 mM, pH 7.4).12 To probe
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2005, 283, 539-543.
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J. S.; Richardson, D. C. Nature (London) 1983, 306, 287-290. (b) Allison,
S. A.; Bacquet, R. J.; McCammon, J. A. Biopolymers 1988, 27, 251-269.
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McCammon, J. A. Methods Enzymol. 1991, 202, 473-497.
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416. (b) Jogl, G.; Rozovsky, S.; McDermott, A. E.; Tong, L. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 50-55.
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Natl. Acad. Sci. U.S.A. 2002, 99, 5018-5023. (b) Hong, R.; Fischer, N.
O.; Verma, A.; Goodman, C. M.; Emrick, T.; Rotello, V. M. J. Am. Chem.
Soc. 2004, 126, 739-743.
(12) (a) You, C.-C.; De, M.; Han, G.; Rotello, V. M. J. Am. Chem. Soc. 2005,
127, 12873-12881. (b) You, C.-C.; De, M.; Rotello, V. M. Org. Lett. 2005,
7, 5685-5688.
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13573.
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A R T I C L E S
You et al.
Figure 2. Generation rate (V) of 4-nitroaniline from different substrates
(2.0 mM) under catalysis by ChT (3.2 µM) or ChT-NP_4EG_Glu complex
(3.2 µM/0.8 µM). Inset shows the normalized activity of ChT-NP complex
to ChT toward different substrates.
Figure 3. Relative activity of ChT-NP_4EG_Glu complex against S1 at
varying NaCl concentrations. The activities were normalized to that of free
ChT at respective salt solutions. Line was added to guide eyes.
increase of salt concentration in a sigmoidal profile, which is
also attributed to the complex disruption at higher salt concen-
trations.
the effect of nanoparticles on ChT activity, a glutamic-acid-
functionalized nanoparticle (NP_4EG_Glu,17 0.8 µM) was first
incubated with ChT (3.2 µM) and the activity of the supramo-
lecular complexes was investigated against different substrates
in the same buffer system. As shown in Figure 2, native ChT
has comparable activities toward the three substrates with diverse
charge characteristics (S1-S3). Upon binding to NP_4EG_Glu,
however, ChT shows distinctly different catalytic behaviors
toward these substrates. For cationic substrate S1, ChT activity
increases by ca. 3-fold. For neutral and negatively charged
substrates S2 and S3, however, ChT activity decreases by ca.
50% and ca. 95%, respectively. These observations demonstrated
that nanoparticle receptors can tune, rather than simply inhibit,
the activity of ChT.
Kinetic data for ChT activity are crucial for understanding
its enzymatic behavior in the presence of amino-acid-function-
alized nanoparticles. It has been well demonstrated that R-chy-
motrypsin-catalyzed hydrolysis of amide substrates is fully
described by the three-step model shown in eq 1.19 First, the
enzyme reversibly binds substrate to give the enzyme-substrate
complex (ES), followed by nucleophilic attack by the active
site serine to form the acyl enzyme (ES′) intermediate and
release the amine product (P1). The amine product for all
substrates used in this study is 4-nitroaniline. Hydrolysis of the
acyl intermediate affords the free enzyme and the carboxylic
acid (P2) which retains the functional group (R1) from the
substrate.
Activity assays were performed at varying salt concentrations
to verify that the changes of enzymatic activity arise from the
formation of protein-particle complexes. In the control experi-
ments, slightly enhanced activity was observed for free ChT
with increasing salt concentrations (ca. 30% in 500 mM NaCl).
Therefore, the activity of the ChT-nanoparticle couple was
normalized to that of free ChT at the same salt concentrations
to eliminate this effect. As shown in Figure 3, the normalized
activity of ChT-NP_4EG_Glu toward substrate S1 decreases
with increasing salt concentrations and reaches a plateau in
NaCl solutions of above 500 mM. We have demonstrated
that the driving force for the ChT-nanoparticle complex
formation is surface complementary electrostatic interaction. The
presence of a large amount of competitive ions would attenuate
such interaction.18 In this context, the observed phenomenon
indicates that the superactivity of ChT-NP_4EG_Glu toward
S1 is related to complex formation. The intrinsic activity of
ChT recovered along with the collapse of the complexes at
elevated salt concentrations. Similarly, the activity of ChT-
NP_4EG_Glu toward anionic substrate S3 increases with the
k2
8
k3
E + S yk1z ES 9E+S′
98 E + P2
(1)
k-1
P1
Applying the steady-state assumption to [ES′], the kinetics
of the hydrolysis reaction can be analyzed in terms of eq 1 to
give an identical form with the Michaelis-Menten equation.
The general equations for velocity (ν0) and catalytic constants
(kcat) and substrate specificity constants (kcat/KM) are shown
below:
kcat[E][S]
V0 )
(2)
(3)
(4)
KM + [S]
k2k3
kcat
)
k2 + k3
k1k2
kcat/KM )
k-1 + k2
The activity of ChT in the absence and presence of various
amino-acid-functionalized nanoparticles was investigated with
varying substrate concentrations to quantify the Michaelis-
Menten parameters. The formation of 4-nitroaniline was linearly
(17) The nanoparticle nomenclature initiates with NP followed by the number
of ethylene glycol units and the three letter code of terminal amino acids.
That is, NP_4EG_Glu represents glutamic-acid-functionalized gold nano-
particle with a tetra(ethylene glycol) tether.
(18) (a) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.;
Academic Press: London, 1992. (b) Verma, A.; Simard, J. M.; Rotello, V.
M. Langmuir 2004, 20, 4178-4181.
(19) Hedstrom, L. Chem. ReV. 2002, 102, 4501-4523.
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Nanoparticle Modulation of Chymotrypsin Behavior
A R T I C L E S
Table 1. Effect of Amino-Acid-Functionalized Gold Nanoparticles
on Kinetic Parameters of R-Chymotrypsina
1
1
substrate
enzyme
k
cat/10-2 s-
K
M/mM
k
cat/KM/M-1 s-
ChT
4.37
5.08
5.63
5.31
16.34
9.32
9.93
5.85
6.70
6.35
16.18
9.29
10.74
8.16
5.40
6.46
5.46
5.40
6.18
0.26
0.29
0.26
0.65
0.24
0.19
0.24
0.74
0.36
0.37
0.22
0.24
0.26
0.63
0.35
0.43
0.26
1.5
67.2
211.7
296.3
221.3
220.8
258.9
268.4
265.9
279.2
244.2
256.8
265.4
249.8
313.8
36.0
9.8
15.6
13.8
74.5
1.3
2.6
2.1
ChT/NP_4EG_Gly
ChT/NP_4EG_Ala
ChT/NP_4EG_Leu
ChT/NP_4EG_Asp
ChT/NP_4EG_Asn
ChT/NP_4EG_Glu
ChT/NP_4EG_Gln
ChT/NP_3EG_Gly
ChT/NP_3EG_Leu
ChT/NP_3EG_Asp
ChT/NP_3EG_Asn
ChT/NP_3EG_Glu
ChT/NP_3EG_Gln
ChT
ChT/NP_4EG_Leu
ChT/NP_4EG_Asp
ChT/NP_4EG_Glu
ChT
ChT/NP_4EG_Leu
ChT/NP_4EG_Asp
ChT/NP_4EG_Glu
S1
S2
S3
6.6
3.5
3.9
0.83
2.0
Figure 4. Substrate saturation curves for the hydrolysis of substrate S1 by
ChT (1.8 µM) in the absence and presence of various amino-acid-
functionalized nanoparticles (0.45 µM) in sodium phosphate buffer (5 mM,
pH 7.4) at 30 °C. The lines represent the best fit of the data using the
Michaelis-Menten equation.
1.1
1.2
a Sodium phosphate buffer solution (5 mM, pH 7.4) at 30.0 °C. [ChT]
) 1.8 µM, [NP] ) 0.45 µM. The experimental errors are within 10%.
dependent on the reaction time during the monitoring period
(15 min) at all the substrate concentrations used. The initial
reaction rate (V0) was thus calculated from the slope of
absorption changes vs the reaction time, where the absorbance
was converted to a concentration scale by a molar absorption
coefficient of 9800 M-1 cm-1 for 4-nitroaniline.20 Typical
substrate saturation curves for the hydrolysis of S1 by ChT and
ChT-nanoparticle complexes are depicted in Figure 4. The solid
lines represent the best fit of the data using eq 2.
From Figure 4, it is seen that the hydrolysis of the substrate
S1 by ChT and ChT-nanoparticle complexes follows the
Michaelis-Menten mechanism. Interestingly, the substrate
saturation curves are dependent on the structure of nanoparticle
side chains. According to the binding constants and stoichiom-
etries obtained previously,12 it is estimated that >90% ChT
molecules are bound to the nanoparticles to afford supramo-
lecular complexes (refer also to Figure S1 in the Supporting
Information) at the concentrations used, i.e., 1.8 µM ChT and
0.45 µM nanoparticles. Therefore, the differences on the
Michaelis-Menten curves are attributed to the complex forma-
tion. Similar phenomena are also observed for the hydrolysis
of substrates S2 and S3. The kinetic parameters kcat, KM, and
kcat/KM were subsequently evaluated from the nonlinear least-
squares curve-fitting analysis using the Origin 7.0 program
(OriginLab Co., Northampton, USA), which are compiled in
Table 1.
It is noted from Table 1 that native ChT affords similar
specificity constants (kcat/KM) for the charged S1 and S3 and a
slightly smaller specificity constant for neutral S2. Upon binding
to the amino-acid-functionalized nanoparticles, the specificity
constants of ChT increase significantly for cationic S1 but
decrease for S2 and S3. We attribute this to electrostatic
interactions between the functionalized nanoparticles and
substrates that affect substrate association with ChT. When
acylation is much slower than substrate release (k-1 . k2), then
kcat/KM ) k2/KS, where KS denotes the dissociation constant
of the ES complex. As nanoparticles bind to the surface of
ChT and the acylation step is largely confined within the
active site, ChT binding to nanoparticles is more likely to
alter the dissociation constant for substrate (KS) than to
affect acylation, k2. For cationic substrate S1, kcat/KM increases
∼4-fold upon ChT binding to the anionic particles, suggesting
that KS has decreased. Similarly, the decrease of kcat/KM for S2
and S3 is attributed to an increasing KS for nanoparticle-bound
ChT.
Although kcat/KM is generally regarded as the “specificity
constant” or “catalytic proficiency”, this value is actually
disconnected from real rates of net product formation and
complete catalytic turnovers. Northrop has suggested that kcat/
KM can be viewed as the ability of an enzyme to capture its
substrates.21 As the substrates differ from each other only by
their charge, it seems that the ability of ChT-nanoparticle
complexes to capture these substrates is highly dependent upon
substrate charge. To understand this phenomenon, the pertinent
structural features of the ChT-nanoparticle complexes need to
be discussed. The nanoparticles associate with ChT through
surface binding, while the active pocket of the enzyme is kept
vacant, allowing the access of suitable substrates. Potential
obstacles to substrate capture are steric hindrance and electro-
static interactions with the anionic monolayer of nanoparticles
near the entrance of the active site (Figure 5a). The electrostatic
attraction between the cationic substrate S1 and the anionic
monolayer facilitates the accumulation of this substrate at the
nanoparticle surface, increasing its local concentration. The
favorable electrostatic attraction results in the facilitated substrate
capture. For neutral substrate S2, there is only a moderate
reduction in substrate capture as there is merely steric hindrance
present. When the anionic substrate S3 is employed, the
electrostatic repulsion between the monolayer and substrate
further reduces the accessibility of the substrate to the enzyme,
leading to the poorest capture. Thus, our results are entirely
(20) Stra¨ter, N.; Sun, L.; Kantrowitz, E. R.; Lipscomb, W. N. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 11151-11155.
(21) Northrop, D. B. J. Chem. Edu. 1998, 75, 1153-1157.
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A R T I C L E S
You et al.
125 once ChT is bound to glutamic-acid-functionalized nano-
particles. When a leucine-functionalized nanoparticle is em-
ployed, an even higher substrate selectivity of 170 for the S1
and S3 couple is obtained. It is necessary to point out that the
kcat/KM values vary only in a small range for a certain substrate
against different ChT-nanoparticle systems due to the com-
pensation effect between kcat and KM (vide post). Nevertheless,
such results demonstrate that monolayer-controlled uptake of
substrates provides a powerful means for enhancing the substrate
selectivity of enzymes.
From Table 1, it is also interesting to note that the catalytic
constant (kcat) of ChT when complexed with nanoparticles is
again related to the charge of substrates. That is, ChT displays
a slight increase in kcat for cationic substrate S1, essentially no
change for neutral substrate S2, and drastically decreases for
anionic substrate S3 in the presence of nanoparticles. As the
local pH for both anionic and cationic substrates in the presence
of a certain nanoparticle should be similar, the observed variation
of kcat values cannot originate from the local pH changes. Since
the catalytic constant is related to the formation (k2) and
decomposition (k3) of the acyl intermediate, nanoparticles could
affect either process. Bender and co-workers have proposed that
acylation is a rate-limiting step for the hydrolysis of amide
substrates while deacylation is rate-determining for the hydroly-
sis of ester substrates of serine proteases, a mechanism that has
gained wide acceptance.23 However, some experiments suggest
that, even in the case of amides, deacylation can be a
rate-limiting step.24
In the current case, we propose that kcat is determined by
product release, a step which is combined with deacylation into
k3. Three separate arguments support this proposal. First, if
ChT-NP binding does not perturb the active site, then acylation
(k2) will be little affected by the presence of the nanoparticles;
any changes in kcat must result from changes in k3. Second, kcat
and kcat/KM vary independently for most of the nanoparticles in
Table 1, indicating that the rate-limiting step for kcat is not found
in the expression for kcat/KM; this excludes k2 as the rate-limiting
step on kcat for all but the fastest ChT-nanoparticle adducts.
Third, a “burst phase” is always observed for the ChT-catalyzed
hydrolysis of the substrates before the reaction levels off to a
slower steady-state rate (Figures S2 and S3 in the Supporting
Information). As turnover was monitored by the release of
4-nitroaniline, such a phenomenon indicates that k3 is the rate-
determining step for the hydrolysis reaction.25 Therefore, the
observed changes of kcat values are mainly attributed to the effect
of nanoparticles on the release of P2. As shown in Figure 5b,
the deacylation process involves not only hydrolysis of the acyl
enzyme but also the diffusion of the second product (P2) from
the active site to regenerate free enzyme. The diffusion process
should be related to the electrostatic interactions between the
charged substrates and the anionic monolayer of the nanopar-
ticles, mirroring the effect seen for substrate binding. For the
cationic substrates, as P2 contains the positively charged tail,
release from the active site appears to be facilitated by the
Figure 5. Electrostatic interaction-controlled (a) uptake of the substrate
and (b) removal of the carboxylate product within the nanoparticle-bound
enzyme.
consistent with the monolayer of nanoparticle acting as a
selectivity filter which either enhances or diminishes substrate
access to the active site.
Electrostatic interactions play a key role in biomacromolecular
binding, an effect that is mimicked in forming the ChT-
nanoparticle complexes. Small-molecule steering by electrostat-
ics is also found in many enzymes, such that charged substrates
bind to the active sites with enhanced affinity. In Cu/Zn SOD
the local positive charge provided by Lys and Arg residues steers
the anionic substrate, O2-•, toward the active site.14a This leads
to a remarkably high rate of turnover (kcat/KM ∼109 M-1 s-1
)
that is partially limited by diffusion. A similar situation is found
in human H-chain ferritin, where electrostatic gradients found
at channels are suggested to guide cations, such as aquated Fe3+
,
entering or leaving the mineralized ferric hydroxide core.22
Acetylcholinesterase has a highly anionic pore leading to the
active site, which engenders rapid turnover of the cationic
substrate acetylcholine.15 The ChT-nanoparticle adducts gain
a similar selectivity filter by virtue of the charged monolayer
from the nanoparticle.
Although the free ChT shows comparable specificity for three
substrates (S1-S3), the kcat/KM values for ChT complexed with
a given nanoparticle always decrease in the order S1 > S2 >
S3 (Table 1). For example, ChT shows a selectivity of 0.9 for
S1 relative to S3, but this value is increased by 140-fold to ca.
(23) (a) Zerner, B.; Bond, R. P. M.; Bender, M. L. J. Am. Chem. Soc. 1964, 86,
3674-3679. (b) Bender, M. L.; Kezdy, F. J.; Gunter, C. R. J. Am. Chem.
Soc. 1964, 86, 3714-3721.
(24) (a) Christenen, U.; Ipsen, H.-H. Biochim. Biophys. Acta 1979, 569, 177-
183. (b) Stein, R. L.; Viscarello, B. R.; Wildonger, R. A. J. Am. Chem.
Soc. 1984, 106, 796-798. (c) Wang, E. C. W.; Hung, S.-H.; Cahoon, M.;
Hedstrom, L. Protein Eng. 1997, 10, 405-411.
(22) Douglas, T.; Ripoll, D. R. Protein Sci. 1998, 7, 1083-1091.
(25) Hartley, B. S.; Kilby, B. A. Biochem. J. 1954, 56, 288-297.
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Nanoparticle Modulation of Chymotrypsin Behavior
A R T I C L E S
particles can assist in the accumulation of cationic substrates
on the nanoparticle surface and subsequently the uptake by the
enzyme.
The sensitivity of kcat to functionality, however, indicates that
a step unique to kcat depends on the surface functionality. The
only such step in the kinetic scheme (eq 1) is the release of P2
(k3). The release of the cationic P2 is enhanced by the more
hydrophilic groups. We can see that the kcat values decrease as
the amino acid side chains become more hydrophobic, irrespec-
tive with the length of the oligo(ethylene glycol) tethers.
Although the kcat values seem to correlate with hydrophobicity,
this may mask the real correlation. From Table 1, we can note
that complexes of ChT with nanoparticles functionalized with
dianionic amino acids such as aspartic acid and glutamic acid
always give the highest catalytic constants. As we have
discussed above, the electrostatic interaction controls the release
of the hydrolyzed product away from the active site. If there
are more negative charges on the monolayer, the electrostatic
attraction would increase P2 release within the charged mi-
croenvironment. Consequently, it is not surprising to see that
the nanoparticles with dianionic amino acids show the highest
kcat values. In this context, the correlation between kcat values
and hydrophobicity indices indeed reflects the correlation of
kcat with the charge of the side chain, as the more charge the
side chain has, the more hydrophilic it is.
Figure 6. Correlation of kinetic parameters of ChT/NP complexes against
substrate S1 to the hydrophobicity index of amino acid side chain in
nanoparticles. Lines were added to guide the eye.
Conclusion
electrostatic attraction to the negatively charged nanoparticles.
By contrast, the negatively charged P2 resulting from anionic
S3 experiences electrostatic repulsion with the anionic mono-
layer, leading to a reduced rate of product release. Consequently,
the observed kcat value for cationic substrates increases whereas
the value for anionic substrates decreases in the presence of
nanoparticles. For the neutral substrate S2, the deacylation
process is not affected by the presence of nanoparticles due to
the absence of charged functionalities. Essentially the same kcat
values are observed for this substrate before and after com-
plexation of ChT with nanoparticles.
For substrates S2 and S3, the catalytic constants of ChT-
nanoparticle do not change significantly with respect to the
amino acid functionalities. However, the systematic investigation
on the hydrolysis of S1 by ChT in the presence of various
nanoparticles revealed that the catalytic behavior is dependent
on the properties of the amino acid side chains. We have
demonstrated previously that the hydrophobicity of amino acid
side chains can affect the binding strength between the nano-
particles and the protein.12 Herein, we plot the enzyme kinetic
constants against the hydrophobicity index26 of amino acid
side chains in the nanoparticles. As can be seen from Figure 6,
the kcat and KM values decrease smoothly according to the
hydrophobicity index, whereas kcat/KM is nearly invariant.
For cationic substrate S1, the different nanoparticle-ChT
complexes afford essentially the same kcat/KM value of 258 (
29 M-1 s-1, irrespective of the hydrophobicity index. If we
view kcat/KM as the ability of the enzyme-particle complexes
to capture the substrate, then nanoparticle functionality does
not impart selectivity in the uptake of the anionic S1 by the
enzyme bound to the nanoparticle surface. This result is
reasonable as the redundant surface carboxylates of the nano-
In summary, we have systematically investigated the enzy-
matic kinetics of ChT upon binding to amino-acid-functionalized
gold nanoparticles toward different substrates and demonstrated
that the complex formation provides a powerful tool to tune
the enzyme specificity. The association of ChT with anionic
nanoparticles leads to the increase of specificity to the positively
charged substrate and the decrease of specificity to the
negatively charged substrate. Such enhanced substrate selectivity
originates from the electrostatic interaction as well as the steric
repulsion of the substrates with the nanoparticle monolayer.
Significantly, the presence of nanoparticles can also affect the
catalytic constants of the enzyme toward different substrates.
The underlying mechanism is attributed to the electrostatic-
interaction-controlled diffusion of the hydrolyzed product. These
results revealed that the inhibition of the enzymes by nanopar-
ticles is far different from that of conventional competitive or
noncompetitive mechanisms. Monolayer-functionalized nano-
particles provide a potent scaffold for creation of an enzyme
modulator based on surface recognition.
Experimental Section
Materials. R-Chymotrypsin (Type I-S from bovine pancreas) and
N-succinyl-L-phenylalaine p-nitroanilide (SPNA) were purchased from
Sigma and used as received. All the other chemicals were obtained
from Aldrich or Fisher unless otherwise stated. Amino-acid-function-
alized gold nanoparticles12 and SPNA derivatives S1-S313 were
synthesized according to the reported procedures. Disodium hydrogen
and sodium dihydrogen phosphate were dissolved in deionized, distilled
water (18 MΩ) to make a 5 mM phosphate buffer solution of pH 7.4,
which was used throughout the enzymatic study.
Activity Assays. Activity assays were performed in sodium phos-
phate buffer (5 mM, pH 7.4) with [ChT] ) 1.8 µM and [NP] ) 0.45
µM and varied substrate concentrations (i.e., 0.05, 0.1, 0.15, 0.2,
0.3, 0.4, 0.5, 0.75, 1.0, 1.5, and 2.0 mM). The enzymatic hydrolysis
reaction was initiated by adding a substrate stock solution (20 µL) in
(26) Monera, O. D.; Sereda, T. J.; Zhou, N. E.; Kay, C. M.; Hodges, R. S. J.
Pept. Sci. 1995, 1, 319-329.
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J. AM. CHEM. SOC. VOL. 128, NO. 45, 2006 14617

A R T I C L E S
You et al.
ethanol to a ChT-NP solution (180 µL). After pre-equilibrium for 3
min, enzyme activity was followed by monitoring product formation
every 15 s for 15 min at 405 nm with a microplate reader (EL808IU,
Bio-Tek Instruments, or SpectraMax M5, Molecular Devices). The
assays were done in duplicate or triplicate, and the averages are reported.
The absorbance was converted to a concentration scale by a molar
extinction coefficient of 9800 M-1 cm-1 for 4-nitroaniline.20 In the
experiments of varying salt concentration, sodium chloride was added
into the phosphate buffer (5 mM, pH 7.4) to make the corresponding
solution.
Acknowledgment. This research was supported by the
NSF Center for Hierarchical manufacturing at the University
of Massachusetts (NSEC, DMI-0531171) and the ONR
(N000140510501).
Supporting Information Available: Three figures (Figures
S1-S3) for activity assays. This material is available free of
charge via the Internet at http://pubs.acs.org
JA064433Z
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14618 J. AM. CHEM. SOC. VOL. 128, NO. 45, 2006