Artificial metalloprotease with active site comprising aldehyde group and Cu(II)cyclen complex

Article abstract of DOI:10.1021/ja052191h

To design artificial proteases that cleave peptide backbones of a wide range of proteins at selected sites, artificial active sites comprising the Cu(II) complex of cyclen (Cu(II)Cyc) and aldehyde group were synthesized on a cross-linked polystyrene. The

Full text of DOI:10.1021/ja052191h

Published on Web 06/14/2005
Artificial Metalloprotease with Active Site Comprising
Aldehyde Group and Cu(II)Cyclen Complex
Sang Ho Yoo, Byoung June Lee, Hyunsook Kim, and Junghun Suh*
Contribution from the Department of Chemistry, Seoul National UniVersity,
Seoul 151-747, Korea
Received April 6, 2005; E-mail: jhsuh@snu.ac.kr
Abstract: To design artificial proteases that cleave peptide backbones of a wide range of proteins at selected
sites, artificial active sites comprising the Cu(II) complex of cyclen (Cu(II)Cyc) and aldehyde group were
synthesized on a cross-linked polystyrene. The aldehyde group was employed as the binding site in view
of its ability of reversible formation of imine bonds with ꢀ-amino groups of Lys residues exposed on the
surface of proteins and Cu(II)Cyc as the catalytic group for peptide hydrolysis. The two polymeric artificial
metalloproteases synthesized in the present study cleaved all of the protein substrates examined (myoglobin,
γ-globulin, bovine serum albumin, human serum albumin, lysozyme, and ovalbumin), manifesting saturation
kinetic behavior. At 50 °C and pH 9.0 or 9.5, Km was (1.3-22) × 10-4 M, comparable to those of natural
proteases, and kcat was (6.0-25) × 10-4 s-1, corresponding to half-lives of 4.6-19 min. Intermediacy of
the imine complexes formed between the aldehyde group of the catalyst and the ꢀ-amino groups of Lys
residues of the substrates was confirmed by the trapping experiment with NaB(OAc)3H. MALDI-TOF MS
of the proteolytic reaction mixtures revealed formation of various cleavage products. Structures of some of
the cleavage products were determined by using carboxypeptidase A and trypsin. Among various cleavage
sites thus identified, Gln(91)-Ser(92) and Ala(94)-Thr(95) were the major initial cleavage sites in the
degradation of myoglobin by the two catalysts. The selective cleavage of Gln(91)-Ser(92) and Ala(94)-
Thr(95) was attributed to general acid assistance in peptide cleavage by Tyr(146) located in proximity to
the two peptide bonds. Broad substrate selectivity, high cleavage-site selectivity, and high proteolytic rate
are achieved, therefore, by positioning the aldehyde group in proximity to Cu(II)Cyc attached to a cross-
linked polystyrene.
Introduction
groups. When organic functional groups are exploited as the
catalytic centers, cooperation of two or more catalytic groups
Designing enzyme-like catalysts for hydrolysis of peptide
bonds of proteins is challenging in view of the high stability1,2
of peptide bonds as well as importance of proteins and peptides
in modern biology and bio-related chemistry. Intramolecular
catalysis of hydrolysis of unactivated peptides by organic
functional groups3 or metal ions4 as well as by the cooperative
action5 of a metal ion and an organic functional group has been
intensively investigated since the 1970s. Intermolecular catalysts
with peptidase activity, however, have been synthesized only
in recent years. Now, several intermolecular catalysts have been
reported for hydrolysis of peptide bonds by using either metal
complexes4-14 or organic functionalities15-19 as the catalytic
is needed. On the other hand, a single metal complex can act
as the catalytic center by playing various catalytic roles.20
Metal complexes capable of site-selective cleavage of proteins
or peptides have been designed in attempts to obtain novel
biochemical reagents or more advanced biomimetic catalysts.
Early studies in this area employed metal complexes in
combination with oxidoreductive additives to achieve either
hydrolytic21 or oxidative22-24 cleavage of the target protein. Site-
(12) Kaminskaia, N. V.; Johnson, T. W.; Kostic, N. M. J. Am. Chem. Soc. 1999,
121, 8663-8664.
(13) Saha, M. K.; Bernal, I. J. Chem. Soc., Chem. Commun. 2003, 612-613.
(14) Kasai, M.; Ravi, R. G.; Shealy, S. J.; Grant, K. B. Inorg. Chem. 2004, 43,
6130-6132.
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8913.
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(4) Sutton, P. A.; Buckingham, D. A. Acc. Chem. Res. 1987, 20, 357-364.
(5) Suh, J.; Park, T. H.; Hwang, B. K. J. Am. Chem. Soc. 1992, 114, 5141-
5146.
(15) Suh, J.; Hah, S. S. J. Am. Chem. Soc. 1998, 120, 10088-10093.
(16) Suh, J.; Oh, S. J. Org. Chem. 2000, 65, 7534-7540.
(17) Oh, S.; Chang, W.; Suh, J. Bioorg. Med. Chem. Lett. 2001, 11, 1469-
1472.
(18) Kim, H.; Paik, H.; Kim, M.-s.; Chung, Y.-S.; Suh, J. Bioorg. Med. Chem.
Lett. 2002, 12, 2557-2560.
(19) Kim, H.; Kim, M.-S.; Paik, H.; Chung, Y.-S.; Hong, I. S.; Suh, J. Bioorg.
Med. Chem. Lett. 2002, 12, 3247-3250.
(6) Chin, J. Acc. Chem. Res. 1991, 24, 145-152.
(7) Suh, J. Acc. Chem. Res. 2003, 36, 562-570.
(8) Chin, J.; Jubian, V.; Mrejen, K. J. Chem. Soc., Chem. Commun. 1990,
1326-1328.
(20) Suh, J. Acc. Chem. Res. 1992, 25, 273-279.
(21) Rana, T. M.; Meares, C. F. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10578-
10582
(9) Zhu, L.; Qin, L.; Parac, T, N.; Kostic, N. M. J. Am. Chem. Soc. 1994, 116,
5218-5224.
(22) Shepartz, A.; Cuenoud, B. J. Am. Chem. Soc. 1990, 112, 3247-3249.
(23) Hoyer, D.; Cho, H.; Schultz, P. G. J. Am. Chem. Soc. 1990, 112, 3249-
3250.
(24) Gallagher, J.; Zelenko, O.; Walts, A. D.; Sigman, D. S. Biochemistry 1998,
37, 2096-2104.
(10) Hegg, E. L.; Burstyn, J. N. J. Am. Chem. Soc. 1995, 117, 7015-7016.
(11) Jang, B.-B.; Lee, K.-P.; Min, D.-H.; Suh, J. J. Am. Chem. Soc. 1998, 120,
12008-12016.
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A R T I C L E S
Yoo et al.
selective hydrolytic cleavage25-28 of peptides or proteins without
the oxidoreductive additives requires highly effective catalytic
centers for hydrolysis of peptide bonds. Several kinds of metal
complexes have been examined for their ability to hydrolyze
peptide bonds,6-14 but most of them had low catalytic rates
unless they were tethered to peptide substrates especially under
physiological conditions.
It was observed that the protein-hydrolyzing activity11 of Cu-
(II) complex of cyclen (Cu(II)Cyc) and DNA-hydrolyzing
activity29,30 of Co(III) complex of cyclen were enhanced
remarkably upon attachment to cross-linked polystyrene. Sub-
sequently, it was attempted to design artificial enzymes for
peptide hydrolysis possessing selectivity for cleavage sites by
using the Cu(II) complexes of cyclen and related ligands as the
catalytic centers and cross-linked polystyrene as the solid
support. To introduce a substrate-recognizing site in proximity
to the catalytic center, an artificial peptidase was prepared by
attaching the guanidinium group close to the Cu(II) complex
of a tetraaza ligand attached to polystyrene.25 In another study,
an active site comprising three proximal Cu(II) centers was
prepared by transfer of a trinuclear metal center confined in a
bowl-shaped molecule to polystyrene.26 Those polystyrene
derivatives selectively hydrolyzed carboxyl-containing amides
by cleaving the peptide bonds adjacent to the carboxyl groups.
The cleavage-site selectivity was attributed to recognition of
the carboxylate anion of the substrate by the carboxylate-binding
group of the artificial active site.
protein substrates (broad substrate selectivity) by cleaving the
substrate at selected positions on the polypeptide backbone (high
site selectivity). To possess both the broad substrate selectivity
and the high site selectivity as digestive proteases or protea-
somes, the artificial proteases should be able to form complexes
with a variety of protein substrates effectively and to cleave
peptide bonds at selected positions in the resulting complexes.
For this purpose, the aldehyde group is employed in the
present study as the binding site of the artificial proteases
because the aldehyde group can form imine bonds with the
ꢀ-amino groups of Lys residues exposed on the surface of
proteins. Since the imine bonds are readily hydrolyzed, the
artificial protease equipped with an active site containing the
aldehyde group may be able to form complexes with a variety
of proteins reversibly. If a catalytic group with high proteolytic
activity is positioned in proximity to the aldehyde group, the
catalytic group may cleave a peptide bond in vicinity to the
Lys residue of the substrate complexed to the artificial protease.
In the present study, polymeric artificial metalloproteases with
active sites comprising Cu(II)Cyc and the aldehyde group were
synthesized. The cooperation between the aldehyde group and
the metal center in the action of the artificial proteases in
cleavage of various proteins is described in this article.
Results
The first artificial protease with selectivity for cleavage site
in the hydrolysis of a protein instead of simple amides was
obtained in a later study:27 the active site was designed by
attachment of a molecular entity containing two cyclens to a
polystyrene derivative to obtain a polymer indicated by [Cyc]2-
PS, followed by addition of Cu(II) ion to the cyclen moieties
to obtain [Cu(II)Cyc]2-PS. In the hydrolytic degradation of
myoglobin by [Cu(II)Cyc]2-PS, two pairs of intermediate
proteins accumulated. Peptide linkages of Gln(91)-Ser(92) and
Ala(94)-Thr(95) were the initial cleavage sites leading to the
formation of the protein fragments. On the basis of a molecular
modeling study, the site selectivity was attributed to anchorage
of one Cu(II)Cyc unit of the catalytic module to a heme
carboxylate of myoglobin. Although high site selectivity for the
initial cleavage of a protein substrate was achieved with the
artificial metalloprotease, the catalyst failed to cleave other
common proteins such as γ-globulin or albumin.
Synthesis of Artificial Metalloproteases. The cyclen-
aldehyde conjugates built on the backbone of polystyrene in
the present study are indicated by A-PS and B-PS. As the
control catalysts lacking the aldehyde groups, Acont-PS and
Bcont-PS were also prepared.
One of the next challenges in the area of synthetic artificial
proteases is to design catalysts that hydrolyze a wide range of
(25) Suh, J.; Moon, S.-J. Inorg. Chem. 2001, 40, 4890-4895.
(26) Moon, S.-J.; Jeon, J. W.; Kim, H.; Suh, M. P.; Suh, J. J. Am. Chem. Soc.
2000, 122, 7742-7749.
The synthetic routes for preparation of A-PS, Acont-PS,
B-PS, and Bcont-PS are summarized in Schemes 1 and 2. The
precursors (A5, A5cont, B1, or B1cont) of catalytic modules were
synthesized by the Ugi condensation reaction. In the Ugi
reaction, one-pot condensation of a carboxylic acid, an aldehyde,
an amine, and an isocyanide produces an N-acyl amino acid
amide.31 As the polymeric support of the artificial proteases, a
(27) Yoo, C. E.; Chae, P. S.; Kim, J. E.; Jeong, E. J.; Suh, J. J. Am. Chem. Soc.
2003, 125, 14580-14589.
(28) Milovic, N. M.; Badjic, J. D.; Kostic, N. M. J. Am. Chem. Soc. 2004, 126,
696-697.
(29) Jeung, C. S.; Kim, C. H.; Min, K.; Suh, S. W.; Suh, J. Bioorg. Med. Chem.
Lett. 2001, 11, 2401-2404.
(30) Jeung, C.-S.; Song, J. B.; Kim, Y.-H.; Suh, J. Bioorg. Med. Chem. Lett.
2001, 11, 3061-3064.
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A R T I C L E S
Scheme 2. Synthetic Route to B-PS and Derivatives
cross-linked polystyrene derivative containing aminomethyl
groups attached to a part of the phenyl rings was used. The
content of aminomethyl group in the polystyrene derivative was
1.5 mmol/g: 17% of the phenyl rings of polystyrene contained
aminomethyl group. After A5, A5cont, B1, or B1cont was attached
to the polystyrene derivative, the primary amines on the polymer
were acetylated, and the protecting groups on the catalytic
modules were removed to obtain A-PS, Acont-PS, B-PS, and
Scheme 1. Synthetic Route to A-PS and Derivatives
Bcont-PS. By the reaction with p-toluenesulfonylhydrazide, the
aldehyde groups of A-PS and B-PS were converted to the
corresponding hydrazones leading to the formation of Ahydraz
-
PS and Bhydraz-PS. By treatment of the cyclen-containing
polymers with CuCl2, Cu(II) ion was complexed to the cyclen
moieties to form Cu(II)A-PS, Cu(II)Acont-PS, Cu(II)Ahydraz
PS, Cu(II)B-PS, Cu(II)Bcont-PS, and Cu(II)Bhydraz-PS.
-
The contents of Cu(II) ion in Cu(II)A-PS, Cu(II)Acont-PS,
Cu(II)Ahydraz-PS, Cu(II)B-PS, Cu(II)Bcont-PS, and Cu(II)-
Bhydraz-PS were measured by inductively coupled plasma
absorption-emission spectroscopy (ICP-AES) after Cu(II) ion
was released from the polystyrene derivatives by multiple
treatment with 1 N HNO3. Based on the amounts of the Cu(II)
ion, the contents of the catalytic modules were calculated as
0.49 mol % (relative to styrene moieties) for Cu(II)A-PS, 0.59
mol % for Cu(II)Acont-PS, 0.47 mol % for Cu(II)Ahydraz-PS,
1.3 mol % for Cu(II)B-PS, 0.90 mol % for Cu(II)Bcont-PS,
and 1.3 mol % for Cu(II)Bhydraz-PS.
Electron probe microanalysis (EPMA) estimated the molar
ratio of Cu(II), S, and Br on the surface of Cu(II)Ahydraz-PS as
1.00:0.81:0.88 and that of Cu(II) and S on the surface of Cu-
(II)Bhydraz-PS as 1.00:0.87, respectively. Considering the pos-
sible nonspecific adsorption of the Cu(II) ion, incomplete
formation of the hydrazones, and/or the experimental error
involved in EPMA measurement, these data are consistent with
the structure of the catalytic modules introduced to Cu(II)A-
PS and Cu(II)B-PS.
Kinetic Measurement. Proteolytic activity of Cu(II)A-PS,
Cu(II)Acont-PS, Cu(II)B-PS, and Cu(II)Bcont-PS was exam-
(31) Domling, A.; Ugi, I. Angew. Chem., Int. Ed. 2000, 39, 3168-3210.
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Figure 1. Results of SDS-PAGE performed on myoglobin (4.9 µM)
incubated with Cu(II)A-PS (Co ) 0.45 mM) at pH 9.5 and 50 °C.
Figure 3. Plot of ko against Co for the hydrolysis of myoglobin catalyzed
by Cu(II)A-PS (b) and Cu(II)Acont-PS (O) at pH 9.5 and 50 °C.
Figure 2. Plot of ln[S]/[S]o against time for the electrophoretic bands
presented in Figure 1. The relative concentration of substrate was measured
by analyzing the density of the electrophoretic bands. The straight line
corresponds to ko of 7.7 × 10-4 s-1
.
ined by using horse heart myoglobin, bovine serum γ-globulin,
bovine serum albumin, human serum albumin, chicken egg
white lysozyme, and chicken egg ovalbumin as the substrates.
Myoglobin is oxidized to metmyoglobin in the presence of
oxygen. The myoglobin purchased from a commercial source
and used in the present study was also in the met form, as
checked by its visible spectrum.32
While the buffer solution containing a protein substrate (1.4-
7.0 µM) was shaken with the resin, disappearance of the protein
was observed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis33,34 (SDS-PAGE). Typical results of SDS-PAGE
performed on the protein substrates cleaved by the polystyrene-
based catalysts are illustrated in Figure 1. That the disappearance
of the electrophoretic bands of the protein substrates was not
due to the adsorption onto the resin was confirmed by measuring
the total amino acid contents of the product solution separated
from the resin according to the method described previously.16
Figure 4. Plot of ko against Co for the hydrolysis of myoglobin catalyzed
by Cu(II)B-PS (b) and Cu(II)Bcont-PS (O) at pH 9.5 and 50 °C.
γ-Globulin has two subunits with distinctly different molecular
weights (25 and 50 kDa). Since the density of the electrophoretic
band of the light chain was weak, the kinetic data were collected
only for the degradation of the heavy chain.
Rate data were collected by varying the amount of the catalyst
(Co). Here, Co is expressed as the concentration of the catalytic
module obtainable when the resin is assumed to be dissolved.
The ko values were measured for each substrate in the presence
of each polystyrene derivative at various pH’s and at fixed Co
and So concentrations under the conditions of Co . So to identify
the optimum pH. At the optimum pH thus selected, the ko values
were measured at various Co concentrations under the conditions
of Co . So. As shown by the dependence of ko on Co (Figures
3, 4, and S1-S10 of Supporting Information), saturation kinetic
behavior was observed for the cleavage of all of the protein
substrates by Cu(II)A-PS and Cu(II)B-PS. Under the same
The rate of protein cleavage at 50 °C was measured by
monitoring the decrease in the intensity of the electrophoretic
bands corresponding to the substrate protein.11,15-19,27 The
pseudo-first-order kinetic constant (ko) was estimated from the
logarithmic plot such as that illustrated in Figure 2. Rate data
were collected by varying the speed of shaking the reaction
mixture containing the polystyrene-based catalyst and the protein
substrate, and it was found that ko reached a plateau value at
the shaking speed of 1200 rpm. Thus, kinetic data were collected
at this shaking speed. The ko values stand for cleavage of the
substrate protein molecule itself and do not provide information
on further fragmentation of the initial cleavage products.
conditions, cleavage of the protein substrates by Cu(II)Acont
-
PS or Cu(II)Bcont-PS was much slower than that by Cu(II)A-
PS and Cu(II)B-PS.
(32) Bodwell, C. E.; McClain P. E. In The Science of Meat and Meat Products,
2nd ed.; Price, J. F., Schweigert, B. S., Ed.; W. H. Freeman: San Francisco,
CA, 1971; p 97.
(33) Lameli, U. K. Nature 1970, 227, 680-685.
(34) Hames, B. D. In Gel Electrophoresis of Proteins; Hames, B. D., Rickwood,
D., Eds.; IRL Press: New York, 1990; Chapter 1.
Kinetic data for the cleavage of the protein substrates by Cu-
(II)A-PS or Cu(II)B-PS can be analyzed in terms of Michae-
lis-Menten scheme (eq 1) as was done with the reactions
catalyzed by other polystyrene-based artificial proteases.11,27
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A R T I C L E S
Table 1. Values of Kinetic Parametersa for the Cleavage of Various Protein Substrates by Cu(II)A-PS and Cu(II)B-PS at 50 °C and at the
Optimum pH
1
1
1 b
substrate
catalyst
pH
k
cat (10-4 s-
)
K
m (10-3 M-
)
k )
cat/Km (s-1 M-
myoglobinc
Cu(II)A-PS
Cu(II)B-PS
Cu(II)A-PS
Cu(II)B-PS
Cu(II)A-PS
Cu(II)B-PS
Cu(II)A-PS
Cu(II)B-PS
Cu(II)A-PS
Cu(II)B-PS
Cu(II)A-PS
Cu(II)B-PS
9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.0
9.5
9.5
9.5
9.5
11
6.7
22
0.32
0.77
1.3
0.57
0.92
1.2
0.79
1.1
0.13
1.3
3.5 (0.005)
8.7 (0.003)
1.7 (0.008)
1.4 (0.005)
0.87 (0.005)
0.73 (0.005)
2.3 (0.008)
0.61 (0.006)
4.8 (0.002)
0.46 (0.002)
2.1 (0.003)
0.68 (0.002)
myoglobin
γ-globulind
γ-globulin
7.8
8.0
8.7
18
6.7
6.2
6.0
25
bovine serum albumind
bovine serum albumin
human serum albumin
human serum albumin
lysozyme
lysozyme
ovalbumin
ovalbumin
1.2
2.2
15
a Parameters kcat and Km become kcat and Kmapp, respectively, when the scheme of eq 5 is used instead of eq 1. b Values in parentheses are kcat/Km
measured with the respective control polymer (Cu(II)Acont-PS or Cu(II)Bcont-PS) under the same conditions. Accuracy of these values is low. c For
[Cu(II)Cyc]2-PS,27 kcat ) 0.95 × 10-4 s-1, Km ) 2.0 × 10-3 M, and kcat/Km ) 0.047 s-1 M-1 at pH 9.0 and 50 °C. d No catalysis in protein degradation
was observed with [Cu(II)Cyc]2-PS.27
app
Under the conditions of Co . [CS], pseudo-first-order kinetic
behavior is expected with ko being derived as eq 2. Kinetic data
illustrated in Figures 3, 4, and S1-S10 were analyzed according
to eq 2 with a nonlinear regression program. The values of kcat,
Km, and kcat/Km for the cleavage of the protein substrates by
Cu(II)A-PS or Cu(II)B-PS are summarized in Table 1. For
Cu(II)Acont-PS or Cu(II)Bcont-PS, ko was proportional to Co,
and the proportionality constant can be taken as kcat/Km (Km .
Co, eq 2). The values of kcat/Km thus estimated for the action of
Cu(II)Acont-PS or Cu(II)Bcont-PS are also summarized in Table
1.
k2
C + S yk1z CS
98 C + P
(1)
k-1
ko ) kcatCo/(Km + Co)
kcat ) k2
(2)
(3)
(4)
Km ) (k-1 + k2)/k1
After Cu(II)A-PS or Cu(II)B-PS was incubated with bovine
serum albumin at various pH’s until the protein was almost
completely degraded, the polymer was recovered and used again
to cleave bovine serum albumin. The activity of the recovered
polymer was almost identical with that of the fresh polymer.
The active sites of the artificial proteases, therefore, are not
appreciably damaged during incubation with the proteins under
the conditions adopted for collection of the kinetic data.
Identification of Cleavage Sites. During incubation of a
protein substrate with a polystyrene-based catalyst, aliquots of
the buffer solution were taken to measure the matrix-assisted
laser desorption/ionization time-of-flight mass spectrum (MALDI-
TOF MS). An example is illustrated in Figure 5, with MALDI-
TOF MS taken at various intervals for the degradation of
myoglobin in the presence of Cu(II)A-PS. Disappearance of
myoglobin, formation and decay of some intermediate proteins,
and accumulation of product proteins are shown in the MALDI-
TOF MS of Figure 5. Each new MALDI-TOF MS peak
represents a cleavage product obtained by the action of the
catalysts.
Figure 5. MALDI-TOF MS obtained during incubation of myoglobin (So
) 4.9 µM) with Cu(II)A-PS (Co ) 0.45 mM) at pH 9.5 and 50 °C. The
peaks labeled with asterisks (m/z ) 16 952 and 8476) are those of
myoglobin. The peaks with m/z of 10 382, 10 089, 6876, 6582, 3984, 3663,
and 2912 are identified as Gly(1)-Ala(94), Gly(1)-Gln(91), Ser(92)-Gly-
(153), Thr(95)-Gly(153), Ser(117)-Gly(153), Gly(1)-Phe(33), and Ser-
(92)-His(116), respectively.
reaction mixture isolated at various time intervals was partially
separated by HPLC to obtain fractions containing fewer numbers
of cleavage products. Each cleavage product contained in the
fraction was subjected to structural analysis by treatment with
carboxypeptidase A or trypsin followed by MALDI-TOF MS
measurement. Carboxypeptidase A releases C-terminal amino
acid residues of a protein in a sequential manner, whereas trypsin
cleaves peptide bonds on the carboxyl side of Lys or Arg.
The sizes of new protein fragments obtained after treatment
of a cleavage product with carboxypeptidase A provide infor-
mation for amino acid sequence at the C-terminus of the
cleavage product as described previously.27,35 When the C-
terminal amino acid of a cleavage product is Gly, Asp, Glu,
Cleavage sites for protein degradation can be identified by
determining the structures of the cleavage products correspond-
ing to the peaks in the MALDI-TOF MS. The content of each
(35) Chae, P. S.; Kim, M.-s.; Jeung, C.-S.; Lee, S. D.; Park, H.; Lee, S.; Suh,
J. J. Am. Chem. Soc. 2005, 127, 2396-2397.
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was incubated for 3 days; 72% of the total amino acid residues
were recovered in the buffer solution.
Discussion
It is well-established that the Cu(II) complex of cyclen
catalyzes cleavage of peptide bonds by hydrolysis: formation
of amino and carboxyl groups upon cleavage of a peptide bond
was confirmed, and catalytic turnover in peptide hydrolysis was
observed.27,37,38
As demonstrated by the kinetic data summarized in Figures
3, 4, and S1-S10 as well as in Table 1, Cu(II)A-PS and Cu-
(II)B-PS effectively degraded all of the six protein substrates
examined in the present study. The values of kcat/Km for
myoglobin degradation by Cu(II)A-PS and Cu(II)B-PS are
20-80 times greater than that by [Cu(II)Cyc]2-PS reported27
previously, as summarized in Table 1. Catalysts Cu(II)A-PS
and Cu(II)B-PS are even more effective for γ-globulin and
bovine serum albumin, since these proteins were not cleaved
by [Cu(II)Cyc]2-PS.27 On the other hand, Cu(II)Acont-PS and
Cu(II)Bcont-PS lacking the aldehyde groups in the active sites
exhibited negligible proteolytic activity. By positioning the
aldehyde group in proximity to Cu(II)Cyc attached to a cross-
linked polystyrene, both broad substrate selectivity and high
proteolytic rate are achieved.
Figure 6. MALDI-TOF MS of the protein fragment with m/z of 6582
(obtained as indicated in Figure 5) after treatment with trypsin (1.0 µM) at
pH 8.0 and 25 °C for 20 min. The m/z values of i, ii, iii, iv, v, and vi are
4316, 4086, 3821, 3370, 2284, and 1885, respectively. The diagram shows
how the structures of the fragments were assigned.
The broad substrate selectivity and high proteolytic rate can
be attributed to the imine formation between the aldehyde group
of the artificial protease and the amino groups of the protein
substrate as schematically indicated by 1. Essentially all globular
proteins possess Lys residues which provide ammonium cations
on the protein surfaces. Imine formation between the Lys-amino
group of the protein substrate and the aldehyde group of the
polystyrene-based artificial protease would anchor the protein
on polystyrene. The broad substrate selectivity manifested by
Cu(II)A-PS and Cu(II)B-PS is attributable to the anchorage
of the protein substrates through imine bonds. Upon formation
of the covalent complex, cleavage of the polypeptide backbone
of the bound protein substrate could occur more readily since
the reaction between the catalytic Cu(II) center and the scissile
peptide bond becomes entropically more favorable.
Pro, or Arg, the cleavage product resists the action of carboxy-
peptidase A. Then, treatment of the cleavage product with
trypsin can provide a different kind of information on the
structure of the cleavage product, as exemplified by the MALDI-
TOF MS illustrated in Figure 6.
The same analysis was performed for the other protein
substrates, but structures were positively identified for fewer
cleavage products compared with myoglobin degradation. This
was because MALDI-TOF MS peaks of the cleavage products
obtained from the other proteins were weaker and broader than
those from myoglobin. The structures of the cleavage products
and the corresponding cleavage sites identified for the proteolytic
actions of Cu(II)A-PSand Cu(II)B-PS are summarized in
Table 2.
Trapping of Imine Intermediate. Since the aldehyde groups
of the artificial proteases can form imine bonds with amino
groups of the protein substrates, attempts were made to trap
the imine intermediates with NaB(OAc)3H, which is known to
reduce imines much faster than aldehydes. Since reductive
amination with NaB(OAc)3H is usually carried out under weakly
acidic conditions36 and Cu(II)A-PS and Cu(II)B-PS are
deactivated under acidic conditions, the trapping experiments
were carried out at pH 6.0. When myoglobin (So ) 4.9 µM)
was degraded by Cu(II)A-PS (Co ) 0.45 mM) at pH 6.0 and
50 °C, ko was 6.8 × 10-6 s-1. After disappearance of myoglobin
was complete, 88% of the total amino acid residues initially
introduced by myoglobin were recovered in the buffer solution.
When 5 mM NaB(OAc)3H was initially introduced to the
reaction mixture, ko was 1.3 × 10-4 s-1 and only 15% of the
total amino acid residues were recovered in the buffer solution.
When the same reaction was carried out first without NaB-
(OAc)3H to complete the degradation of myoglobin, NaB-
(OAc)3H was added to the degradation mixture, and the mixture
The effect of imine formation can be analyzed quantitatively
in terms of a modified Michaelis-Menten scheme of eq 5. Here,
CS stands for the noncovalent intermediate formed between the
(37) Jeon, J. W.; Son, S. J.; Yoo, C. E.; Hong, I. S.; Song, J. B.; Suh, J. Org.
Lett. 2002, 4, 4155-4158.
(38) Jeon, J. W.; Son, S. J.; Yoo, C. E.; Hong, I. S.; Suh, J. Bioorg. Med. Chem.
2003, 11, 2901-2910.
(36) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah,
R. D. J. Org. Chem. 1996, 61, 3849-3862.
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9598 J. AM. CHEM. SOC. VOL. 127, NO. 26, 2005

Site-Selective Artificial Metalloproteases
A R T I C L E S
Table 2. Cleavage Products and the Corresponding Cleavage Sites Identified for the Proteolytic Action of Cu(II)A-PS and Cu(II)B-PS
substrate
cleavage product
cleavage site
myoglobina
Gly(1)-Phe(33)b,c
Gly(1)-Gln(91)b,c
Gly(1)-Ala(94)b,c
Ser(92)-His(116)b
Ser(92)-Gly(153)b,c
Thr(95)-Gly(153)b,c
Ser(117)-Gly(153)b
Asp(1)-Gln(33)b
Phe(33)-Thr(34)
Gln(91)-Ser(92)
Ala(94)-Thr(95)
Gln(91)-Ser(92), His(116)-Ser(117)
Gln(91)-Ser(92)
Ala(94)-Thr(95)
His(116)-Ser(117)
bovine serum albumina
human serum albumina
lysozymea
Gln(33)-Cys(34)
Ile(141)-Trp(213)b,c
Gly(206)-Val(230)b
Asp(1)-Ile(25)b
Gln(140)-Ile(141), Trp(213)-Ser(214)
Phe(205)-Gly(206), Val(230)-Thr(231)
Ile(25)-Ala(26)
Asp(1)-Gln(33)b
Gln(33)-Cys(34)
Met(298)-Met(329)c
Ser(91)-Arg(114)b,c
Gly(104)-Arg(125)b,c
Gly(104)-Leu(129)b
Glu(297)-Met(298), Met(329)-Phe(330)
Ala(90)-Ser(91), Arg(114)-Cys(115)
Asn(103)-Gly(104), Arg(125)-Gly(126)
Asn(103)-Gly(104)
a The numbers of amino acid residues contained in the protein molecules are 153 for myoglobin, 583 for bovine serum albumin, 585 for human serum
albumin, and 129 for lysozyme. b Identified for the cleavage by Cu(II)A-PS. c Identified for the cleavage by Cu(II)B-PS.
substrate and the catalyst, CS′ the imine formed between the
substrate and the catalyst, and CP1 the imine formed between
the cleavage product and the catalyst. By applying steady-state
approximation to [CS] and [CS′], rate parameters are derived
as eqs 6-8 under the condition of Co . So for the disappearance
of S. The same saturation kinetic behavior (eqs 2 and 6) is
predicted for the dependence of ko on Co by both eqs 1 and 5.
(k-2/k2)Kd; eq 10) can be attributed to the high stability (k-2/k2
, 1) of the imine complex (CS′) compared with that of the
noncovalent complex (CS).
The intermediacy of the imine complex is further supported
by the trapping experiment using NaB(OAc)3H. The rate for
the disappearance of myoglobin in the presence of Cu(II)A-
PS was raised by 19 times upon addition of NaB(OAc)3H under
the conditions described in the Results section. The enhanced
rate for disappearance of the protein substrate is consistent with
trapping of the imine intermediate with the borohydride reagent.
A major portion of the amino acid residues originally contained
in the protein substrate was recovered after completion of the
proteolytic reaction in the absence of NaB(OAc)3H. When NaB-
(OAc)3H was added after completion of the proteolysis, most
of the amino acid residues were still recovered. On the other
hand, addition of NaB(OAc)3H in the initial stage of the
proteolysis by Cu(II)A-PS resulted in immobilization of most
of the amino residues on the polystyrene surface. This again
supports trapping of imine intermediate by NaB(OAc)3H.
k3
9
C + S yk1z CS yk2z CS′
8 CP1 + P2 yk4z C + P1 + P2 (5)
k-1
k-2
k-4
ko ) kcatappCo/(Kmapp + Co)
(6)
(7)
kcatapp ) k2k3/(k2 + k-2 + k3) e k3
Kmapp ) (k2k3 + k-1k3 + k-1k-2)/k1(k2 + k-2 + k3) (8)
kcatapp ) k3 (when k3 , k-2 , k2)
(9)
Kmapp ) k-1k-2/k1k2 ) (k-2/k2)Kd , Kd
The structures of the seven cleavage products and the
positions of four cleavage sites were identified for the action
of Cu(II)A-PS on myoglobin. The four cleavage sites are
indicated on the X-ray crystallographic structure40 of horse heart
metmyoglobin in Figure 7. Among the seven cleavage products,
five were also identified in the action of Cu(II)B-PS. It is
certain that Gln(91)-Ser(92) and Ala(94)-Thr(95) are among
the initial cleavage sites since the two protein fragments (Gly-
(1)-Gln(91) and Ser(92)-Gly(153); Gly(1)-Ala(94) and Thr-
(95)-Gly(153)) obtained by the cleavage at each of the two
sites were positively identified. Figure 5 does not reveal any
other large fragment arising from another possible initial
cleavage site.
(when k3 , k-2 , k2 , k-1) (10)
When the kinetic scheme is modified to include more
intermediates, the expressions of kcat (or kcatapp) and Km (or Km
app
)
become more complex. Under special circumstances, the
physical meaning of kcatapp and Kmapp represented by eqs 7 and
8 is clearer. It is likely that the rate-determining step is the
peptide-cleaving step (k3 , k-2). In view of the broad substrate
selectivity and high catalytic rates observed with Cu(II)A-PS
or Cu(II)B-PS, it is reasonable to assume that [CS′] is much
higher than [CS] (k-2 , k2). One may assume that the CS
complex is in fast equilibration with C and S (k2 , k-1 and,
app
Myoglobin contains 19 Lys residues which are marked with
K in Figure 7. With the whole structure of myoglobin including
the side chains of Lys residues being frozen, molecular
mechanics calculations were carried out by using molecular
modeling programs (HyperChem and Sculpt) to identify the
potential anchorage sites leading to the cleavage at Gln(91)-
Ser(92) or Ala(94)-Thr(95). With the aldehyde groups of the
thus, Km ) Kd ) k-1/k1). Under these conditions, kcat and
app
Km can be approximated as eqs 9 and 10.
The values of kcatapp are (6.0-25) × 10-4 s-1, corresponding
to half-lives of 4.6-19 min. Since k3 stands for the rate constant
app
for peptide cleavage in CS′ to form CP1, kcat
represents
effectiveness of the catalytic center in peptide cleavage. Under
the identical conditions, the half-life for the spontaneous
hydrolysis of peptide bonds is 10-30 years.1,2 The values of
(39) Polga´r, L. Mechanisms of Protease Action; CRC Press: Boca Raton, FL,
1989.
(40) Evans, S. V.; Brayer, G. D. J. Mol. Biol. 1990, 213, 885-897.
app
Km are (1.3-22) × 10-4 M, being comparable to the Km
values of proteolytic enzymes.39 The low values of Km ()
app
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J. AM. CHEM. SOC. VOL. 127, NO. 26, 2005 9599

A R T I C L E S
Yoo et al.
Figure 8. Locations of the scissile peptide bonds of Gln(91)-Ser(92) and
Ala(94)-Thr(95) as well as the phenol group of Tyr(146) in the X-ray
crystallographic structure of myoglobin.
Scheme 3. Possible Action of Tyr-146 as a General Acid Catalyst
Figure 7. Backbone of the X-ray crystallographic structure of metmyo-
globin. In the center is the heme group. Red arrows indicate the cleavage
sites: A for Phe(33)-Thr(34), B for Gln(91)-Ser(92), C for Ala(94)-
Thr(95), and D for His(116)-Ser(117). Lys residues are marked with K
followed by numerals.
catalyst linked to the amino group of a Lys residue, whether
the attack of the Cu(II)Cyc moiety of the catalyst at the peptide
group of Gln(91)-Ser(92) or Ala(94)-Thr(95) is feasible was
examined. The modeling programs indicated that the anchorage
at one of the 11 Lys resides marked in red and with * in Figure
7 did not involve appreciable steric strain for the attack of the
Cu(II)Cyc at either Gln(91)-Ser(92) or Ala(94)-Thr(95). This
was mainly due to the flexibility of the artificial active site
comprising Cu(II)Cyc and the aldehyde group. If the flexibility
of the protein, especially the long side chains of the Lys residues,
is taken into consideration, it is more difficult to identify the
anchorage site.
peptide cleavage at Gln(91)-Ser(92) and Ala(94)-Thr(95).
Examination of the crystallographic structure of myoglobin
suggested that the phenol group of Tyr(146) may promote the
cleavage of both Gln(91)-Ser(92) and Ala(94)-Thr(95) by
acting as an extra catalytic group. As illustrated in Figure 8,
the distance between the phenol oxygen of Tyr(146) and the
amide nitrogen atom of Ser(92) or Thr(95) is 6.7 or 6.4 Å,
respectively, in the crystallographic structure. Little steric strain
is imposed, however, when the distance between the phenol
oxygen atom and either of the two amide nitrogen atoms is
reduced to 3 Å.
As summarized in Scheme 3, the Cu(II) center may act as a
Lewis acid catalyst to polarize the carbonyl group of the scissile
peptide bond and facilitate attack by hydroxide ion at the
carbonyl carbon. Alternatively, the Cu(II)-bound hydroxide ion
may act as the nucleophile. In either case, the rate-determining
step would be the expulsion of amine moiety from the
tetrahedral intermediate, which requires protonation of the amine
nitrogen by the specific acid or a general acid.20 The phenol of
Tyr(146) may act as the general acid catalyst to accelerate the
rate-determining amine expulsion. At pH 9.0-9.5, the optimum
pH for the polystyrene-based artificial proteases, the phenol of
Tyr is the strongest acid among the organic functionalities
present in the protein molecule.
It is not possible to identify the Lys residue of myoglobin
that is anchored on the aldehyde group of the catalytic module
prior to the initial peptide cleavage. The catalytic center,
however, selectively attacks peptide bonds of Gln(91)-Ser(92)
or Ala(94)-Thr(95) despite the flexibility of the artificial active
site comprising Cu(II)Cyc and the aldehyde group. Interestingly,
Gln(91)-Ser(92) and Ala(94)-Thr(95) were also the initial
cleavage sites in the degradation of myoglobin by [Cu(II)Cyc]2-
PS investigated in the previous study.27 As noted above, the
site selectivity in the action of [Cu(II)Cyc]2-PS was attributed
to anchorage of one of the two Cu(II)Cyc units of the active
site by the heme carboxylate of myoglobin followed by the
attack by the other Cu(II)Cyc unit at the scissile peptide bond.
In the action of [Cu(II)Cyc]2-PS, why Gln(91)-Ser(92) and
Ala(94)-Thr(95) were selectively cleaved among several pep-
tide groups having access to the catalytic center was unanswered.
The initial cleavage of myoglobin at Gln(91)-Ser(92) or Ala-
(94)-Thr(95) involves anchorage of the substrate onto the
polystyrene-based artificial protease by reversible formation of
the imine complexes as well as cleavage of peptide bonds by
the Cu(II)Cyc moiety of the artificial protease with general acid
assistance from the Tyr phenol group provided by the substrate.
The selective cleavage of Gln(91)-Ser(92) and Ala(94)-
Thr(95) in the action of Cu(II)A-PS, Cu(II)B-PS, and [Cu-
(II)Cyc]2-PS despite the structural differences in the catalytic
modules implicates that some structural features may assist the
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9600 J. AM. CHEM. SOC. VOL. 127, NO. 26, 2005

Site-Selective Artificial Metalloproteases
A R T I C L E S
The optimum pH of 9.0-9.5 reflects the overall efficiency of
complexation of the protein by the catalyst, the formation of
imine intermediates, and the catalytic action of Cu(II)Cyc
combined with hydroxide ion and the Tyr phenol group.
structure, the artificial proteases would become more effective
and achieve higher reaction rates and high amino acid specific-
ity.
Experimental Section
Figure 5 indicates that Gly(1)-Gln(91) and Gly(1)-Ala(94)
are degraded rapidly, whereas Ser(92)-Gly(153) and Thr(95)-
Gly(153) are more stable during incubation with Cu(II)A-PS.
Those protein fragments contain Lys residues which can interact
with the aldehyde group of Cu(II)A-PS. The faster degradation
of Gly(1)-Gln(91) and Gly(1)-Ala(94) suggests the presence
of extra catalytic groups provided by the protein fragments,
although no information is available for the three-dimensional
structure of Gly(1)-Gln(91) and Gly(1)-Ala(94).
When myoglobin or its degradation products are reductively
linked to polystyrene by treatment with NaB(OAc)3H, the
amount of total amino acids released to the buffer solution is
considerably reduced, indicating suppression of cleavage of the
polypeptide backbones. The aldehyde group of the polystyrene-
based protease forms imine complexes with various Lys amino
groups of the protein substrate reversibly. In the absence of NaB-
(OAc)3H, various forms of the imine complexes would revers-
ibly transform into one another until the productive forms
undergo protein cleavage. In the presence of NaB(OAc)3H,
however, reduction of the unproductive forms of the imine
complexes can immobilize the proteins blocking the protein
fragmentation.
Preparation of Catalysts. The derivative of poly(styrene-co-
divinylbenzene) with 17% of styryl residues aminomethylated (1.5
mmol NH2 per gram resin) and with 2% cross-linkage was purchased
from Fluka. A solution of a precursor (A5, A5cont, B1, or B1cont) of the
catalytic module or the control module (0.73 mmol) mixed with 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) (280 mg; 0.73 mmol), di-i-propylethylamine (DIEA) (0.40
mL; 2.3 mmol), and N-hydroxybenzotriazole (HOBT) (89 mg; 0.66
mmol) in 5 mL of N,N-dimethylformamide (DMF) was added to the
suspension of the polystyrene (4.0 g) in 30 mL of DMF. The suspension
was degassed for 30 min and shaken at 45 rpm and room temperature
for 1 day. The product resin (A5-PSo, A5cont-PSo, B1-PSo, or B1cont
-
PSo) was collected by filtration, washed with CH2Cl2 (30 mL × 5) and
CH3OH (30 mL × 5), and dried in vacuo. The amino groups of the
resulting resin were acetylated by shaking the resin (4.0 g) with acetic
anhydride (5.7 mL) and DIEA (10 mL) dissolved in 30 mL of DMF at
room temperature for 1 day. The product resin was collected by
filtration, washed with DMF (30 mL × 5) and CH3OH (30 mL × 5),
and dried in vacuo to obtain the acetylated derivative (A5-PS, A5cont
-
PS, B1-PS, or B1cont-PS). Kaiser test43 indicated that the remaining
amino groups were quantitatively acetylated. The acetylated resin (4.0
g) was shaken in the mixture of 6 mL of trifluoroacetic acid (TFA)
and 30 mL of CH2Cl2 at 45 rpm and room temperature for 1 h. The
product resin was collected by filtration and washed with CH2Cl2 (30
mL × 5). Then the resin was shaken in 6 mL of DIEA and 24 mL of
CH2Cl2 at 45 rpm and room temperature for 1 h. The product resin
was collected by filtration, washed with CH2Cl2 (30 mL × 5) and CH3-
OH (30 mL × 5), and dried in vacuo to obtain A-PS, Acont-PS, B-PS,
or Bcont-PS. To the suspension of A-PS or B-PS (0.10 g) in CH3OH
(1.5 mL) were added acetic acid (15 µL) and p-toluenesulfonhydrazide
(2.6 mg, 0.014 mmol). The resulting mixture was shaken at 45 rpm
and room temperature for 5 h. The resin was filtered, washed with
CH3OH (10 mL × 5) and CH2Cl2 (30 mL × 5), and dried in vacuo to
obtain Ahydraz-PS or Bhydraz-PS. To a 0.23 M CuCl2‚2H2O solution in
DMF (25 mL), the cyclen-containing polystyrene derivative (4.0 g)
was suspended, and the resulting mixture was shaken at 45 rpm and
room temperature for 1 day and washed with DMF (30 mL × 5) and
CH3OH (30 mL × 5) to obtain the Cu(II) complex of the cyclen-
containing polystyrene derivative (Cu(II)A-PS, Cu(II)Acont-PS, Cu-
(II)Ahydraz-PS, Cu(II)B-PS, Cu(II)Bcont-PS, or Cu(II)Bhydraz-PS).
Measurements. In kinetic measurements, the shaking speed and
temperature were controlled with a VORTEMP manufactured by
Labnet. pH measurements were carried out with a Dongwoo Medical
DP-880 pH/Ion meter. The degree of cleavage of proteins was measured
by SDS-PAGE with a Mighty Small II SE 250 model. Densities of the
electrophoretic bands were analyzed with a AlphaImager 2200 model
and a AlphaEase model. MALDI-TOF MS analysis was performed with
a Voyger-DE STR Biospectrometry Workstation model. NMR spectra
were recorded with a Bruker DPX 300 MHz model, and the UV-vis
spectra with a Beckman DU 68 UV-vis spectrometer. ICP-AES
measurement was carried out with a Shimadzu ICPS-1000IV model.
EPMA was performed with a CAMECA SX-57 model. Distilled and
deionized water was used for preparation of buffer solutions. Buffers
(0.05 M) used in this study were acetate (pH 5.0), 2-(N-morpholine)-
ethanesulfonate (pH 6.0), N-2-hydroxyethylpiperazine-N′-ethansulfonate
(pH 7.0-8.0), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonate
(pH 8.5), and boric acid (pH 9.0-10.5). Kinetic measurements and
trapping experiments were carried out in the presence of 0.3 M NaCl.
In vivo, digestive proteases and proteasomes mostly hydrolyze
unfolded or precleaved proteins. For example, pepsin cleaves
proteins unfolded in the acidic medium of stomach producing
protein fragments, which are cleaved further by other proteases
in the intestine. Proteasomes, the main enzymes of the non-
lysosomal pathway of protein degradation in cells of higher
organisms, cleave cellular proteins unfolded with ATP.41 In the
industrial application of artificial proteases, however, it is
desirable to hydrolyze a variety of proteins without addition of
denaturing agents. Hydrophilic residues such as ammonium or
carboxylate ions are exposed on the surface of undenatured
globular proteins.42 To synthesize artificial proteases recognizing
undenatured protein molecules, we chose to design recognition
sites targeting the ammonium groups exposed on the surface
of protein substrates. As we expected, artificial proteases Cu-
(II)A-PS and Cu(II)B-PS cleaved all of the protein substrates
tested in the present study by using both the aldehyde and the
Cu(II)Cyc moiety in the active site. Furthermore, the artificial
proteases manifested high cleavage site selectivity.
The immobile artificial proteases can overcome thermal,
chemical, and mechanical instabilities of natural proteases.
Broad substrate selectivity, high proteolytic rate, and high
cleavage-site selectivity are the three major objectives in
designing artificial proteases applicable to protein industry. By
introducing an aldehyde group in proximity to the Cu(II)Cyc
attached to polystyrene, remarkable improvement has been
achieved in those three major goals. By incorporating more
catalytic elements to the artificial active site with a better-defined
(41) Kisselev, A. F.; Goldberg, A. L. Chem. Biol. 2001, 8, 739-758.
(42) It has been recently reported28 that an artificial peptidase based on a Pd-
(II)-cyclodextrin conjugate recognized the phenyl residue of an oligopep-
tide and hydrolyzed the adjacent peptide bond of the oligopeptide. This
catalyst would not cleave the peptide backbones of globular proteins if the
phenyl residues are not exposed on the surface of the protein substrates.
(43) Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal. Biochem.
1981, 117, 147-157.
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A R T I C L E S
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All buffer solutions were filtered with 0.45 µm Millipore microfilter
and autoclaved before use in the kinetic measurements. Horse heart
myoglobin, bovine serum γ-globulin, bovine serum albumin, human
serum albumin, chicken egg white lysozyme, chicken egg ovalbumin,
bovine carboxypeptidase A, and bovine trypsin were purchased from
Sigma and used without further purification. The resins were suspended
in a buffer solution and swollen for 1 h prior to kinetic measurement.
pioneered the area of synthetic macromolecules with enzyme-
like activities.
Supporting Information Available: Experimental procedures
for synthesis of A5, A5cont, B1, and B1cont and plots of ko against
Co (Figures S1-S10) for hydrolysis of γ-globulin, bovine serum
albumin, human serum albumin, lysozyme, and ovalbumin by
the polystyrene-based catalysts. This material is available free
of charge via the Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by Korea
Research Foundation Grant (KRF 2004-041-C00220). This
article is dedicated to the late Prof. Irving M. Klotz, who
JA052191H
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9602 J. AM. CHEM. SOC. VOL. 127, NO. 26, 2005