Voltage-assisted peptide synthesis in aqueous solution by α-chymotrypsin immobilized in polypyrrole matrix

Article abstract of DOI:10.1021/ja950020e

Potential-assisted enzymatic peptide synthesis in aqueous solution has been performed by α-chymotrypsin immobilized in conducting polymer polypyrrole matrix. Chymotrypsin is electrochemically adsorbed on a Pt electrode, and then a thin polypyrrole membran

Full text of DOI:10.1021/ja950020e

1
824
J. Am. Chem. Soc. 1996, 118, 1824-1830
Voltage-Assisted Peptide Synthesis in Aqueous Solution by
R-Chymotrypsin Immobilized in Polypyrrole Matrix
Golam Faruque Khan, Eiry Kobatake, Hiroaki Shinohara,
Yushihito Ikariyama, and Masuo Aizawa*
Contribution from the Department of Bioengineering, Tokyo Institute of Technology,
Nagatsuta, Midori-ku, Yokohama 226, Japan
X
ReceiVed January 3, 1995
Abstract: Potential-assisted enzymatic peptide synthesis in aqueous solution has been performed by R-chymotrypsin
immobilized in conducting polymer polypyrrole matrix. Chymotrypsin is electrochemically adsorbed on a Pt electrode,
and then a thin polypyrrole membrane is electrochemically prepared on the enzyme layer. The enzyme/polypyrrole
on an electrode retains chymotrypsin activity and the equilibrium of the enzymatic reaction shifts to the synthesis by
ca. 25% due to the hydrophobic environment caused by polypyrrole matrix. Here, Ac-Phe-OEt and Ala-NH2 are
used as model substrates of acyl donor ester and a nucleophile acceptor, respectively, to synthesize Ac-Phe-Ala-
NH2. Both the synthetic and the hydrolytic activity of the immobilized chymotrypsin are found to be enhanced
several times when a positive potential (0.4-0.8 V vs Ag/AgCl) is applied to the electrode. In addition, the equilibrium
of the enzymatic reaction shifts further to synthesis. About 73% of synthetic yield of Ac-Phe-Ala-NH2 is obtained
from equimolar concentration (200 mM) of Ac-Phe-OEt and Ala-NH2 in aqueous solution. The effects of solution
pH, temperature, organic solvent concentration, and substrates concentration on the peptide synthesis are also described.
An explanation of possible effects of the applied voltage which causes the remarkable increase of catalytic activity
is also presented.
Introduction
Scheme 1. Reaction Mechanism of
R-Chymotrypsin-Catalyzed Peptide Synthesis
Proteases are well-known to work for hydrolyzing peptide
bond. However, the reverse reaction, i.e., peptide synthesis can
also be catalyzed by proteases, which was first reported by
1
Bergmann and Fraenkel-Conrat in 1937. They reported the
papain-catalyzed amide bond synthesis. Since then, many
reports on the reverse reaction of proteases2
-18
have appeared.
Although all proteases are theoretically capable of catalyzing
their synthetic and hydrolytic reactions, the unfavorable equi-
librium in aqueous solution, where water is not only a solvent
but is also an excessive reactant, rules out significant function
in the reverse synthesis for most of these enzymes. However,
extensive reversal of enzymatic hydrolysis should be possible
by decreasing the water content in a mixed solvent, which results
in shifting of the equilibrium of a hydrolytic reaction to the
synthesis of peptides.
*
To whom all correspondence should be addressed.
Abstract published in AdVance ACS Abstracts, February 1, 1996.
X
(
20.
1) Bergmann, M.; Fraenkel-Conrat, H. J. Biol. Chem. 1937, 119, 707-
7
The reaction catalyzed by R-chymotrypsin (serine protease,
(
2) Hess, G. P. Enzymes 2nd Edn. 1971, 3, 213-248.
2
EC.3.4.21.1) proceeds in two steps as illustrated in Scheme 1.
(3) Homanberg, G. A.; Mattis, J. A.; Laskowski, M. Biochemistry 1978,
1
7, 5220-5227.
The first step is the coupling of an acyl donor to the serine-195
residue of the enzyme, and the second is a subsequent
competitive deacylation with water or with a nucleophilic
acceptor, such as peptide and C-protected amino acid. In the
former case a hydrolytic product is obtained, whereas in the
later case a peptide is produced. Therefore, peptide synthesis
is favorable in a less aqueous environment at high concentration
of nucleophilic acceptors from the viewpoint of equilibrium of
the reaction. The main drawbacks of the enzymatic peptide
synthesis in water-organic mixed solvent is the susceptibly of
enzymes to denature which may cause very low enzyme activity.
In addition, for the convenient separation of the products the
enzyme catalyst should be immobilized to a solid matrix for
repeated used.
(
(
(
(
(
(
(
4) Morihara, K.; Oka, T. J. Biochem. 1981, 89, 385-395.
5) Morihara, K.; Oka, T. Biochem. J. 1977, 163, 531-542.
6) Nilson, K.; Moshach, K. Biotechnol. Bioeng. 1984, 26, 1146-1154.
7) West, J. B.; Wong, C. H. J. Org. Chem. 1986, 51, 2728-2735.
8) Bergmann, M.; Fruton, J. S. J. Biol. Chem. 1938, 124, 321-329.
9) Behrens, O. K.; Bergmann, M. J. Biol. Chem. 1939, 129, 587-602.
10) Isowa, Y.; Ohmori, M.; Ichikawa, T.; Kurita, H.; Sato, M.; Mori,
K. Bull. Chem. Soc. Jpn. 1977, 50, 2762-2765.
11) Isowa, Y.; Ohmori, M.; Sato, M.; Mori, K. Bull. Chem. Soc. Jpn.
977, 50, 2766-2772.
12) Isowa, Y.; Ichikawa, T.; Ohmori, M. Bull. Chem. Soc. Jpn. 1978,
1, 271-276.
13) Saltman, R.; Vlach, D.; Luisi, P. L. Biopolymers 1977, 16, 631-
38.
(
1
5
6
1
(
(
(14) Luisi, P. L.; Saltman, R.; Vlach, D.; Guarnaccia R. J. Mol. Catal.
977, 2, 133-138.
(
(
(
15) Oka, T.; Morihara, K. J. Biochem. 1977, 82, 1055-1062.
16) Oka, T.; Morihara, K. J. Biochem. 1978, 84, 1277-1283.
17) Tsuzuki, H.; Oka, T.; Morihara, K. J. Biochem. 1980, 88, 669-
To overcome these, a novel approach for peptide synthesis
in an aqueous solution has been performed by taking advantages
of hydrophobic environment in the polypyrrole matrix wherein
chymotrypsin is entrapped. We have reported earlier that the
6
75.
(18) Ferjancic, A.; Puigserver, A.; Gsertner, H. Appl. Microbiol. Bio-
technol. 1990, 32, 651-657.
0
002-7863/96/1518-1824$12.00/0 © 1996 American Chemical Society

Voltage-Assisted Peptide Synthesis
J. Am. Chem. Soc., Vol. 118, No. 8, 1996 1825
A positive potential of 0.55 V was applied for electrochemical
adsorption of the enzyme for 10 min. Then, the enzyme adsorbed
electrode was transferred to another solution containing 0.1 M pyrrole
in a deoxygenated electrolyte whose pH was adjusted beforehand to
5
-6 with STA. The pH was not adjusted when KCl was used as an
electrolyte. After 2 min, a potential of 0.6-0.7 V was applied to
polymerize pyrrole on the enzyme adsorbed electrode. The polymer-
ization was terminated after a defined amount of electricity was passed.
Then, the enzyme immobilized electrode (PP/CT/Pt) was thoroughly
washed with water and kept in water until further experiment.
Peptide Synthesis of Chymotrypsin-Immobilized PP Electrode.
Unless otherwise specified, the reaction mixture was composed of 8
mM acyl donor ester (Ac-Phe-OEt) and 40 mm nucleophilic acceptor
(Ala-NH
2 2 3 3
) in 0.1 M Na CO /NaHCO buffer which contained 2%
acetonitrile for the solubilization of ester substrates. A mixed solvent
of acetonitrile and water with a mixing ratio of 1:1 was used to dissolve
the ester substrates. Finally, the solution was mixed with an aqueous
solution of the nucleophilic acceptor (1 M) and then diluted with the
carbonate buffer solution to make the final concentration. The volume
for the potential-assisted synthesis was 2 mL. All the experiments were
carried out at room temperature (22 ( 2 °C). The potential assisted
synthesis was performed for 60 min.
Figure 1. Preparation of CT-immobilized PP (PP/CT/Pt) electrode.
activity of flavoenzyme,19 PQQ enzyme,20 and NAD enzyme21
immobilized into polypyrrole matrix can be controlled electro-
chemically. These enzymes have electroactive prosthetic groups
and coenzyme. Potential-dependent controllability of these
oxidoreductases’s activity promotes us to study the effects of
potential application on enzymes which have neither electron
transfer path nor electrochemically active cofactor. We have
chosen the well studied protease, R-chymotripsin, since the
mechanism of chymotrypsin has been clarified in details, and
it is one of enzymes of commercial importance.
In the case of peptide synthesis at a higher ester concentration, Ac-
Phe-OEt (4 M) and Ala-NH
2
(2 M) were separately desolved in 100%
ACN and a carbonate buffer of pH 10, respectively. These two
substrates were then mixed in the same buffer solution. Ac-Phe-OEt
was hardly dissolved in the solution, when the concentration is over
ca. 50 mM. Therefore, peptide synthesis was performed in an ester
suspended solution.
A three electrodes system consisting of a PP/CT/Pt working
electrode, a Pt plate counter electrode, and a Ag/AgCl reference
electrode was set in a 10 mL test tube containing the reaction mixture.
Then, a potential was applied for a desired time under magnetically
stirred solution. After or during potential application a 25 µL sample
was directly injected to HPLC for the product analysis. All datum are
the mean of 3-5 experiments.
Assessment of the Hydrophobicity of the Polypyrrole Matrix.
The hydrophobicity in the polypyrrole matrix was assessed from the
fluorescence change of 8-ANS.22 8-ANS was entrapped in PP matrix
during polymerization of pyrrole. 8-ANS (20 mM) was dissolved in
a deoxygenated aqueous solution containing 0.1 M NDADS and 0.1
M pyrrole, and polymerization was performed on a 1.4 cm × 3 cm Pt
plate electrode at 0.7 V with a polymerization charge of 50 mC/sq‚-
cm. After thorough washing with distilled water, the PP/ANS/Pt
electrode was placed into a 1 cm × 1 cm cuvette containing a carbonate
buffer solution (CBS) of pH 9.0. The electrode was positioned so as
to excite the fluorescent probe at an angle of 45°. The resulting light
emission was then detected without changing the position of the
photomultiplier. A wire type counter electrode and a reference electrode
connected with a salt bridge were set the other side of the PP/ANS/Pt
electrode. Fluorescent change was measured by changing applied
potential.
To demonstrate the potential-assisted peptide synthesis, we
have selected a peptide synthesis process by taking Ac-Phe-
OEt and Ala-NH2 as an acyl donor ester and a nucleophile
acceptor, respectively, to synthesis Ac-Phe-Ala-NH2 as reported
by Nilsson and Mosbach.6 We introduced a new hydrophobic
interface, polypyrrole matrix, for the enzyme immobilization.
Here, polypyrrole plays dual roles as a hydrophobic matrix and
a conductive interface as illustrated in Figure 1. If the
hydrophobicity of the interface material is electrochemically
controlled, one can expect that the chymotrypsin-catalyzed
peptide synthesis is remarkably enhanced. In addition, we
speculate that not only the hydrophobicity can be controlled by
the application of a potential but also the surrounding of the
enzyme and its active center is controllable. The potential
application may change the morphology of PP matrix which
may change substrates diffusion and the change of the matrix
pH which certainly have some effects on the enzyme activity.
Experimental Section
Materials. R-Chymotrypsin (Type II: from bovine pancreas, 3X
crystallized, 39 U/mg solid), N-acetylphenylalanine ethyl ester (Ac-
Phe-OEt), and alaninamide (Ala-NH ) were purchased from Sigma.
2
Sodium p-toluenesulfonate (STS), sodium p-toluenesulfonic acid (STA),
naphthalene-1,5-disulfonic acid disodium salt (NDADS), and 8-anilino-
HPLC Analysis. The products were analyzed using a reverse phase
5
HPLC column of Wakosil- C18
.
The eluent, a mixed solvent of
PO /H PO :20 mM),
phosphate buffer solution of pH 2.0 (NaH
2
4
3
4
acetonitrile, and methanol with a ratio of 65:25:10 was pumped through
the column at a flow rate of 1 mL/min at 30 °C. Every component
was monitored spectrophotometrically at 260 nm. Ester substrate,
acidic, and peptide product were baseline separated in all cases. The
peptide yield was calculated from the ratio of the peak area compared
with pure reagent.
Apparatus. Chymotrypsin adsorption, pyrrole polymerization, and
potential-assisted synthesis were performed with a HA-301 potentiostat/
galvanostat of Hokuto Denko Co. (Tokyo), and the electricity for
polymerization was controlled with a HF-201 Coulomb/amperometer
of Hokuto Denko Co.
1
-naphthalene sulfonic acid (8-ANS) were from Kanto Chemical Co.
Pyrrole was purchased from Tokyo Kasei (Tokyo) and was further
distilled before use. Acetonitrile (ACN) and methanol of HPLC grade
were purchased from Wako Pure Chemical. Other chemicals were
guaranteed reagents grade.
Preparation of r-Chymotrypsin-Immobilized Polypyrrole Ma-
trix. Chymotrypsin (40 mg) was dissolved in 2 mL of phosphate buffer
(
20 mM) solution of pH 7.0. A conventional three electrodes system
consisting of a 0.5 cm × 0.5 cm Pt plate working electrode
electrochemically cleaned), a Pt plate counter electrode, and a Ag/
AgCl reference electrode was inserted in this chymotrypsin solution.
(
(
19) Yabuki, Y.; Shinohara, H.; Aizawa, M. J. Chem. Soc., Chem.
Commun. 1989, 14, 945-946.
20) Khan, G. F.; Shinohara, H.; Ikariyama, Y.; Aizawa, M. Anal. Chem.
992, 64, 1254-1258.
21) Yabuki, S.; Shinohara, H.; Ikariyama, Y.; Aizawa, M. J. Electroanal.
Chem. 1990, 277, 179-187.
Results
(
Chymotrypsin-Catalyzed Peptide Synthesis in Solution.
Both the synthetic and the hydrolytic activities of free chymot-
rypsin were investigated in a series of water-acetonitrile mixed
1
(

1
826 J. Am. Chem. Soc., Vol. 118, No. 8, 1996
Khan et al.
Table 1. Effect of Electrolyte on the Activity of R-Chymotrypsin
Immobilized in PP Matrixa
polymerization
potential
synthetic
total
%age of
electrolyte V vs Ag/AgCl yield, mM yield, mM synthetic yield
KCl
STS
NDADS
0.7
0.6
0.6
2.40
3.00
2.98
6.67
7.82
7.61
36
38
39
a
All the reactions were conducted in carbonate buffer of pH 9.0 for
8 h. Reaction volume was 2 mL, and the polymerization charge was
0 mC/cm .
1
1
2
Figure 2. Organic solvent dependent peptide synthesis by free enzyme.
The concentration of CT was 40 µM. Acetonitrile was mixed with
0
.1 M CBS of pH 9.0 before used. The reactions were performed for
2
h and were terminated by the addition of 100 µL of acetic acid: (O)
synthetic product, (0) total product, (b) percentage of synthetic yield.
solution containing 8 mM acyl donor and 40 mM nucleophilic
acceptor. The concentration of CT was 40 µM. Acetonitrile
was mixed with 0.1 M CBS of pH 9.0 before used. The reaction
was performed for 2 h and were terminated by the addition of
Figure 3. Effect of PP membrane thickness on the activity of the PP/
CT/Pt electrode. The reactions were conducted in CBS of pH 9.0 for
1
00 µL of acetic acid. The result is presented in Figure 2. Up
1
8 h. Reactions volume was 2 mL: (O) synthetic product, (0) total
to 20% of ACN, the conversion of the donor ester to products
was completed. Above this content yield gradually dropped to
nearly 50% in a mixed solvent of 80% ACN. Around the 40%-
product, (b) percentage of synthetic yield.
morphology of the polypyrrole membrane changes considerably
when the molecular size of the electrolyte and the polymerization
potential is changed: the electrolyte of larger molecular size is
favorable for the polymerization of loose polymer and polym-
erization at higher potential also produces loose polymer. Since
the biocatalyst is involved, it is preferable to apply a lower
potential to cause less damage to the enzyme. In the case of
the enzyme entrapment the choice of electrolyte is the key step
for controlling matrix morphology. Table 1 shows the effect
of three electrolytes on the PP/CT/Pt electrode activity. In the
case of KCl polymerization was performed at 0.7 V, because it
was the lowest potential at which electropolymerization was
most smoothly performed. As the molecular size of NDADS
and STS and bulkier than KCl, less dense polymer was prepared.
Therefore, the substrate diffusion becomes easier, causing higher
enzyme activity. By taking advantages of bulkier electrolyte
not only easily diffusible polymer was formed but less extreme
potential could be applied for the polymerization, which was
indispensable for the electrochemical immobilization of en-
zymes. Although, both STS and NDADS showed almost same
activity, we employed NDADS as an electrolyte for the
following study.
5
0% of ACN both the synthetic, and the hydrolytic yield were
exceptionally low probably due to the inactivation of the
enzyme. During the experiment it was observed that the enzyme
was completely soluble in the water/ACN mixture up to 50%
of ACN, which might caused the enzyme inactivation. On the
contrary, when the acetonitrile content was above ca. 60%, the
enzyme commenced to be a suspended state. In this suspended
state the enzyme retained its activity as reported by Dastoli et
3
0-32
al.
Therefore, chymotrypsin activity again increased at
around 60% of ACN due to the suspended active enzyme. With
the increase of ACN content the percentage yield of synthetic
product (the ratio of synthetic product to total products)
increased gradually from 15 at 2% of ACN to 81 at 80% of
ACN. This result suggests that the equilibrium of the chymot-
rypsin catalyzed reaction shifts from the hydrolysis to the
synthesis with the increase of the hydrophobic environment,
i.e., with the decrease of water concentration.
Enzyme Activity of Immobilized Chymotrypsin. In the
20,23
previous study,
it was observed that application of a positive
potential (0.5 V) favored very fast adsorption of fructose
dehydrogenase on a Pt electrode. Here, we followed the same
technique of electrochemical adsorption of chymotrypsin on Pt
electrode. We observed that the optimum potential of chymot-
rypsin adsorption was 0.55 V in a phosphate buffer solution of
pH 7.0. The amount of chymotrypsin adsorbed on the electrode
and the hydrolytic activity of the adsorbed chymotrypsin
Figure 3 shows the relation of the membrane thickness to
the enzyme activity. In general, polypyrrole membrane thick-
ness is proportional to polymerization charge. With the increase
in polymerization charge, both the synthesis and the hydrolysis
activities increased up to a polymerization charge of 4-5 mC/
(
measured by the hydrolysis of benzoyl-L-tyrosine ethyl ester
2
cm and then gradually decreased. It seems likely that initial
2
at pH 7.8 at 25 °C) were determined to be 3.5 ( 0.5 µg/cm
and 45 ( 5 mU/cm , respectively. After adsorption, the
increase of activity was caused by the physical stabilization of
2
adsorbed chymotrypsin with PP coating. Therefore, at least 4-5
enzyme-adsorbed electrode was transferred to an electrolyte
solution which contained pyrrole and electrolyte. And then, a
very thin PP matrix was prepared to cover the adsorbed enzyme
as shown in Figure 1.
2
mC/cm polymerization charge was necessary to prepare a
membrane thick enough to avoid enzyme leaking out. The PP
membrane thickness prepared by passing a charge of 4-5 mC/
2
cm corresponds roughly to 10-12 nm. Further increase of
The activity of this enzyme electrode depends on the
morphology and the thickness of polypyrrole membrane. The
PP thickness the enzyme activity gradually decreased due to
the increasing diffusion barrier for the substrates. On the other

Voltage-Assisted Peptide Synthesis
J. Am. Chem. Soc., Vol. 118, No. 8, 1996 1827
Figure 5. Effect of pH on the potential-assisted peptide synthesis by
the PP/CT/Pt electrode: pH 7-8, phosphate buffer; pH 9-12, CBS;
applied potential, 0.6 V; (O) synthetic product and (b) percentage of
synthetic yield.
Figure 4. Potential dependence on the activity of the PP/CT/Pt
electrode: (O) synthetic product, (0) total product, (b) percentage of
synthetic yield.
hand, the percentage of synthetic yield increased with the incrase
in polymerization charge up to 10-12 mC, and, finally became
saturated. This fact suggests that the polypyrrole matrix
provides hydrophobic environment around the entrapped en-
zyme. In the case of soluble chymotrypsin the synthesis was
only 15% at 2% of ACN solution as shown in the Figure 2.
Because of the immobilization of the enzyme in PP matrix the
synthesis increased to ca. 40% in the same 2% of ACN solution.
Therefore, PP matrix is very useful to enhance hydrophobicity
for peptide synthesis in an aqueous solution. In the following
Optimization of the Potential-Assisted Peptide Synthesis.
It is well-known that enzymatic peptide synthesis by CT
effectively proceeds in an alkaline solution. The extent of pH
is also dependent on the type of acyl donor substrate. For
example, in the case of N-protected acid as an acyl donor, the
1
7
optimum pH for the peptide synthesis is around 7.
However,
in the case of ester substrate the synthesis is favored at higher
6
,7,15
pH in the range of pH 9-11.
Most of the ester substrate
is highly unstable at a pH higher than 10 due to the autohy-
drolysis of ester. So, there is a limitation of using the higher
pH buffer for peptide synthesis.
2
experiments, a polymerization charge 10 mC/cm was passed
for immobilizing chymotrypsin.
Figure 5 shows the pH dependence on the potential-assisted
Potential-Assisted Peptide Synthesis. Figure 4 shows the
potential dependence on the synthetic and total yield of the PP/
CT/Pt electrode catalyzed reaction. The rest potential of the
PP/CT/Pt electrode in a buffer solution of pH 9.0 was around
synthesis of Ac-Phe-Ala-NH . With the increase of pH of the
reaction mixture the synthetic yield increased up to pH 10 and
2
then decreased. Ac-Phe-OEt is an unstable ester due to the
autohydrolysis to Ac-Phe-OH at a pH higher than 10. Therefore,
the drop of synthesis product at a pH higher than 10 was mainly
due to the decrease of ester concentration. The percentage of
synthetic yield also increased with the increase of pH from 10%
at pH 7 to 53% at pH 10 and then decreased sharply due to the
autohydrolysis of the ester to Ac-Phe-OH. If the autohydrolyzed
AC-Phe-OH (determined by separate experiment) was deducted
from the product, the percentage of synthesis yield became rather
saturated (dotted line). These results have a good agreement
0
.2 V. At the rest potential, the activity of the PP/CT/Pt
electrode was the minimum. The increase of potential in
negative direction caused a gradual increase of both the synthetic
and the hydrolytic yield up to -0.4 V, and then very slow
decrease was observed. At around -0.4 V both the activities
increased nearly twofold as compares to those of the rest
potential. However, the synthesis as well as the hydrolysis
sharply increased due to the application of a positive potential
and attained the maximum value at around 0.4 V. This
maximum value which is several fold (6-8) higher than that
of at the rest potential continued with a very slow decrease up
to 0.8 V, and then a sharp drop of activity was observed at a
potential higher than 1.0 V. At an extreme potential like 1.0 V
the enzyme might possibly be denatured; therefore, activity was
dropped. The synthetic yield was about 38% at the rest
potential. At a negative potential, the maximum synthesis of
ca. 44% was obtained at -0.4 V, whereas at a positive potential
it was ca. 50% at 0.4 V and decreased to 44% at 0.8 V.
Therefore, application of potential, especially positive potential
between 0.4 V to 0.8 V caused not only the increase of
chymotrypsin activity several times but also caused the shift of
the thermodynamic equilibrium of the enzymatic reaction toward
synthesis. Although the maximum activity and the synthetic
yield (%) was obtained at the potential of 0.4 V, we used 0.6 V
as a most effective potential for peptide synthesis in the
following experiments, because, the standard deviation of yield
was higher at 0.4 V.
5
,6
with the reported results in literature.
From Figure 5, it is
understood that the optimum pH for Ac-Phe-Ala-NH2 synthesis
is pH 10. In the following experiments pH 10 was used.
The effect of the concentration of acetonitrile on the synthesis
of Ac-Phe-Ala-NH is presented in Figure 6. The activity of
2
the PP/CT/Pt electrode was observed to be limited in aqueous
solution (<20%). At higher ACN content synthesis as well as
hydrolysis decreased to nearly negligible at 30% of ACN.
Therefore, the effect of organic solvent on the potential-assisted
synthesis was very severe. The main reason of this lower
activity seems to be the deactivation of the enzyme due to the
combined hydrophobic environment of the organic solvent and
the PP matrix. The inactivation of the enzyme (CT) was not
totally irreversible. At least 50% of its activity was found to
be regained when the electrode was transferred to an aqueous
solution after 2 h incubation in 30% of ACN solution. On the
other hand, the percentage of synthetic yield increased with the
increase of ACN concentration, although the increase rate was
much slower than the free enzyme (Figure 2). Despite the

1
828 J. Am. Chem. Soc., Vol. 118, No. 8, 1996
Khan et al.
Figure 8. Effect of substrates concentration on the potential-assisted
peptide synthesis by the PP/CT/Pt electrode. The concentration of the
nucleophilic acceptor was 5 times more than an acyl donor.
Figure 6. Effect of organic solvent on the potential-assisted peptide
synthesis by the PP/CT/Pt electrode: ACN was mixed with 0.1 M CBS
of pH 10 before used: applied potential, 0.6 V; (O) synthetic product
and (b) percentage of synthetic yield.
rypsin. However, we believe that the application of potential
changes the surrounding of the enzyme and its active site. We
can summarize all the possible changes caused by the application
of a potential as follows: (1) the hydrophobicity of the PP matrix
is changed, (2) pH inside the matrix is changed which may alter
the enzyme conformation to a favorable configuration, (3) PP
matrix is swollen due to the charge interaction, and thus pore
size is increased which favors substrates diffusion, and (4)
relative permeability of the less hydrophobic nucleophilic
acceptor over a more hydrophobic acyl donor into the matrix
is enhanced. We speculate that all of these changes in the
surrounding have some effects on the enzyme activity. To
clarify these effects, we performed a couple of experiments.
We investigated the relationship between the hydrophobicity
of polypyrrole matrix and the applied potential by using a
hydrophobic fluorescence probe, 8-ANS. The highest fluores-
cence intensity of the probe was observed at a negative potential
of -0.4 V and the lowest one at positive potential of 0.6 V. In
every case a significant fluorescence intensity was observed as
shown in Figure 9A, which indicates that whatever the potential
the environment in the PP matrix is hydrophobic, and the
application of the potential changes the matrix hydrophobicity.
Therefore, it can be concluded that the hydrophobicity inside
the polypyrrole matrix depends on the doping state. The doped
anions may have some influence on the change of hydrophobic-
ity; however, in the present study, we did not examine the effect
of anions on the change of hydrophobicity. At the undoped
state (< -0.4 V) the hydrophobicity increases due to the
expulsion of the doped anion. At the doped state (>0.4 V) the
matrix becomes less hydrophobic due to the increase of anion
concentration. Therefore, the potential application caused the
drastic change in fluorescence, depending on an applied potential
as shown in Figure 9B. The initial fluorescent intensity was
gradually restored when the applied potential was terminated.
Although the hydrophobicity of the PP matrix can be controlled
by the potential application, the positive potential is not
necessarily favorable for the peptide synthesis. However, the
peptide synthesis was mostly promoted at potential from 0.4 to
Figure 7. Effect of temperature on the potential-assisted peptide
synthesis by the PP/CT/Pt electrode: (O) synthetic product and (b)
percentage of synthetic yield.
higher synthetic yield (%) at the higher ACN content, the
potential-assisted synthesis should be performed in aqueous
solution.
Figure 7 shows the effect of temperature on the potential
assisted peptide synthesis. The maximum synthetic yield was
obtained at a temperature between 20 and 25 °C, i.e., at room
temperature. At a high temperature (over 30 °C) the yield
became very low. In addition, the percentage of the synthetic
yield decreased gradually with the increase of temperature.
Therefore, the optimum temperature for peptide synthesis is at
lower temperature between 10 and 25 °C.
The percentage of the synthetic yield increased sharply with
the increase of substrates concentration and became nearly
saturated over a concentration of 100 mM of acyl donor as
shown in Figure 8. Eighty-six percent of synthetic yield was
obtained at a concentration of 200 mM of acyl donor. The shift
of the thermodynamic equilibrium toward synthesis was due
mainly to the increase of nucleophilic acceptor. In this
experiment, fivefold excess of nucleophilic acceptor was used.
However, we also performed the experiment with equimolar
concentration of both of the substrates (not shown here). A
significant synthesis yield of 73% was observed at a concentra-
tion of 200 mM. These results indicate a great improvement
in peptide synthesis, because most of the reports claimed that a
several fold excess of nucleophilic acceptor is necessary for the
0
.8 V. This contradiction indicates that other factors including
hydrophobicity should be considered to clarify the enhancement
the peptide synthesis.
There is no direct experimental evidence that application of
potential causes the change of pH inside the PP matrix.
However, it can be easily understood that at a positive potential
6,7,17,18
sufficient synthesis.
-
2-
as anions such as OH and (CO3) doped into the matrix, the
pH is certainly increased. The reverse phenomenon was
Discussion
2
4
observed by Sawai et al. just outside the polyaniline matrix.
We think that this change of pH around the enzyme certainly
has some effects on the conformation of the enzyme active site,
Due to the absence of redox cofactor in the active center there
is no direct effect on the potential on the activity of chymot-

Voltage-Assisted Peptide Synthesis
J. Am. Chem. Soc., Vol. 118, No. 8, 1996 1829
Figure 9. Potential dependence on the hydrophobicity of the PP matrix in terms of fluorescence intensity: (A) fluorescence spectrum at Ex-315
nm and (B) time response at Ex-315 nm and Em-515 nm.
which may lead to the enhancement of synthetic yield as many
groups reported that the synthesis is enhanced at alkaline
pH.
5.0. The deacylation of EP2 would be certainly slower than
the above value in a solution of pH 9-10. Therefore, the rate
limiting deacylation of EP2 can be accelerated simply by
increasing the concentration of nucleophilic acceptor. Results
are shown in Figure 10.
6
,7,15,17
To investigate the kinetics of the enzyme catalyzed peptide
synthesis, a technique for quantifying the efficiency of nucleo-
philes in a kinetic approach is used to examine the reaction of
acyl-enzyme intermediate with nucleophiles25 as presented in
eq 1. The efficiency of a nucleophile is determined by as shown
in eq 2 where [H] and [P] are the final concentrations of the
hydrolysis product and peptide product, respectively, and [N]
is the nucleophile concentration. Determination of p for a given
nucleophile at various concentrations allows for the calculation
of a partition ratio for nucleophile as well as an affinity constant
of the nucleophile for the enzyme. Knowing these parameters
of a nucleophile for the free enzyme and for the immobilized
enzyme, the changes in active-site geometries, which may have
direct responsibility on the synthesis reaction, can be examined.
The ratio of the acid to peptide product can be expressed as
30
the following:
[H]
k
3
′[N] + k
k5[N]
3
′ KN
p
[N]
)
)
(3)
[P]
This equation can be rewritten as in eq 4
k3′[N]
k3′KN
p )
+
(4)
k5
k5
A plot of p vs [N] will yield a line of slope k3′/k5 and a y
intercept of k3′KN/k5, where KN is very similar to a Km for a
normal enzyme reaction. When k5 , k-4,KN is roughly equal
to k-4/k4, the dissociation constant for the EP2‚N complex.
Therefore, KN value is a good parameter to use for comparison
enzyme affinity for nucleophile and for comparison of the
geometries of the S1′ binding site, which in kinetic controlled
synthesis is the most important factor influencing the yield of
P1
k4[N]
k–4
k5
E + S
EP2
EP2•N
E + peptide (P)
(1)
k3[H2O] = k′3′
E + P2 (hydrolytic product: H)
[
H]
P]
p
(2)
31
peptide. In this study, KN value for the free enzyme and for
[
[N]
the immobilized enzyme were calculated to 48 and 214 mM,
respectively. Which strongly suggests that a significant change
in the shape of the S1′ binding site occur due the immobilization
of the enzyme in PP matrix. KN, value for the potential-assisted
synthesis could not be calculated as experimental data did not
yielded a straight line rather yielded a parabolic line. We
speculate that the concentration of the substrates in the PP matrix
is changed when a potential is applied.
In eq 1, the substrate S is acetyl-L-phenylalanine ethyl ester,
the first product P1 is EtOH, and the second product P2 is acetyl-
L-phenylalanine. The acyl-enzyme intermediate is EP2, in which
it is believed that the oxygen of Ser-195 is linked to the carboxyl
carbon of acetyl-L-phenylalanine. In eq 1, k1, k2, k3, and k5 are
the rate constants pertaining to the liberation of P1, P2, and
peptide, respectively.
Gutfreund and Sturlevant26,27 suggested that during the
chymotrypsin-catalyzed hydrolysis of specific substrate esters
the deacylation of acyl-enzyme intermediate (EP2) is the rate
limiting step (k2 . k3). Same results was also published by
Figure 10 shows the effect of the nucleophilic acceptor on
the synthetic and the hydrolytic yields of the three different
systems: (A) free enzyme, (B) PP/CT/Pt electrode without
(
26) Gutfreun, H.; Sturtevant, J. M. Biochem. J. 1956, 63, 656.
(27) Gutfreun, H.; Sturtevant, J. M. Natl. Acad. Sci. U.S.A. 1956, 42,
719.
28) Zerner, B.; Bond, R. P. M.; Bender, M. L. J. Am. Chem. Soc. 1964,
2
8
29
Zerner et al. Hinoe et al. have succeeded to measure the
value of k2 and k3 as 13/s and 2.2/s in a buffer solution of pH
(
(
(
22) Turner, D. C.; Brand, L. Biochemistry 1968, 7, 3381.
23) Khan, G. F.; Shinohara, H.; Ikariyama, Y.; Aizawa, M. J. Elec-
86, 3674-3679.
(29) Himoe, A.; Brandt, K. G.; DeSa, R. J.; Hess, G. P. J. Biol. Chem.
1969, 244, 3483.
troanal. Chem. 1991, 315, 263-273.
24) Sawai, T.; Shinohara, H.; Ikariyama, Y.; Aizawa, M. J. Electroanal.
Chem. 1990, 283, 221-230.
25) West, J. B.; Hennen, W. J.; Lalonde, J. L.; Bibbs, J. A.; Zhong, Z.;
Meyer, Z. L.; Wong, C. H. J. Am. Chem. Soc. 1990, 112, 5313-5320.
(
(30) Riechman, L.; Kasche, V. Biochem. Biophys. Res. Commun. 1984,
120, 686-691.
(31) Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967,
27, 157-162.
(

1
830 J. Am. Chem. Soc., Vol. 118, No. 8, 1996
Khan et al.
the reaction of acyl-enzyme intermediate with water or nucleo-
philic acceptor. In such a situation, it is expected that the
synthesis is enhanced due to the increase of nucleophile
concentration.
When a potential, especially a positive potential, is applied a
significantly higher synthesis is observed [Figure 10C] at a lower
nucleophile concentration and the synthesis becomes relatively
saturated at higher nucleophile concentration as compare to the
synthesis at without potential [Figure 10B]. These results
suggest that the potential is most effective at lower concentration
of nucleophile which indirectly indicates that the application
of potential causes an enhanced diffusion of relatively hydro-
philic nucleophile. We believe that the ratio of nucleophile to
acyl donor in the PP matrix is higher than the bulk solution
when a positive potential is applied. For this reason, the
potential assisted system did not yield a straight line when p vs
[N] was plotted. Based on the above discussion it can be
concluded that all the four points mentioned earlier contributed
to the enhancement of the peptide synthesis.
Conclusion
A significant percentage (ca. 86%) of synthesis yield and a
remarkable enhancement of activity was obtained in aqueous
solution by application of a positive potential (0.4-0.8 V).
Authors believe that this finding will open a new era in
enzymatic peptide synthesis especially in designing an activity
controllable bioreactor for the synthesis of physiologically and
commercially important peptides because it has overcome
several problems for peptide synthesis. The advantage of this
developed system are as follows: (1) it works in aqueous
solution, (2) the enzyme immobilized in PP matrix is more stable
at higher pH, (3) the enzyme-electrode can be repeatedly used,
(4) the activity enhances several times due to the application
Figure 10. Effect on nucleophilic and acceptor: (A) free enzyme, (B)
the PP/CT/Pt electrode at without potential, and (C) the PP/CT/Pt
electrode at 0.6 V. In all the cases, the concentration of acyl donor
of potential, (5) it works with equimolar concentration of acyl
donor and nucleophilic acceptor and (6) catalytic activity can
be controlled by electrochemical means. Solubility of most of
the ester substrate is very low in aqueous solution which is the
main limitation of this system. This problem can be partially
overcome by conducting synthesis in suspended solution.
(Ac-Phe-OEt) was 8 mM in pH 9.0 CBS. [(A) CT 0.22 µM; time, 5
min; (B) time, 2 h; reaction volume; 1 mL; (C) time, 60 min; reaction
volume; 2 mL].
We have explained the mechanism of potential-assisted
peptide synthesis. We have shown that due to the immobiliza-
tion of CT in PP matrix the reaction mechanism is changed.
The acylation step becomes extraordinarily fast when the
deacylation step becomes a solely rate determining step. KN
value increases from 48 mM for free enzyme to 214 mM for
the immobilized enzyme which indicates that the geometry of
the nucleophile binding site is changed to have higher affinity
for nucleophile. The potential application changes the enzyme
surrounding. It causes drastic change in hydrophobicity of the
PP matrix. It increases the diffusion of both substrates and,
especially, the nucleophilic one as the matrix pore size increases
and the hydrophobicity decreases. As a result, the synthesis
increases several times due to the combined effect.
potential application, and (C) PP/CT/Pt electrode at 0.6 V. In
the case of free enzyme [Figure 10A], the synthetic yield
increased sharply at the lower nucleophile concentration and
then continued slow increase with the increase of nucleophile.
While the hydrolytic yield decreased. Total yield increased at
the lower nucleophile concentration and trended to the saturation
at higher concentration. This is quite in agreement with its KN
of 48 mM.
However, the effect of nucleophilic acceptor on the activity
of PP/CT/Pt electrode is different [Figure 10B]. It shows that
with the increase of nucleophilic acceptor the synthetic yield
increased continuously, while the hydrolytic yield remained
unaffected. This is to be expected for reactions in which the
acylation step (k2) is extraordinarily fast and the product yield,
product nature, etc. is determined by the deacylation step, i.e.,
JA950020E