Download Endoproteinase pro-C-catalyzed peptide bond

Endoproteinase Pro-C-catalyzed
peptide bond formation in
frozen aqueous systems
Marion Haensler and Hans-Dieter Jakubke
Faculty of Biosciences, Pharmacy, and Psychology, Institute of Biochemistry, Leipzig University,
Leipzig, Germany
The capability of endoproteinase Pro-C from Flavobacterium meningosepticum to form peptide bonds in frozen
aqueous systems was investigated. In coupling of Bz-Gly-Pro-OMe with various amino acid amides, free amino
acids, and dipeptides, freezing-induced yield enhancement strongly depended on the nucleophilic amino
component ranging from 3–59%. From the different influence of freezing, it can be concluded that freeze
concentration is not the only cause of yield enhancement in frozen aqueous systems.
Endoproteinase Pro-C proved to be a suitable catalyst in Pro-Leu bond formation but its scope of capability
in peptide synthesis is limited. © 1998 Elsevier Science Inc.
Keywords: Endoproteinase Pro-C; Flavobacterium meningosepticum; peptide bond; frozen aqueous system
Introduction
Due to the outstanding stereo- and regiospecificity of
enzymes, protease-catalyzed peptide bond formation has
proved to be an attractive alternative to chemical methods.1,2 Proteases catalyze peptide synthesis under mild
reaction conditions and without time-consuming side chain
protection strategy. Proline is an essential part of many
biologically active peptide sequences which are involved in
regulatory, protective, and destructive processes such as
immunoregulation, coagulation, inflammation, and microbial and viral infections3; therefore, effective synthesis of
proline-containing peptides is of great interest.
Enzyme-catalyzed formation of Pro-X bonds remains
problematic, however, because most proteolytic enzymes do
not cleave peptide bonds including proline. There are only
few reports about the use of proline-specific enzymes in
peptide synthesis. Besides dipeptidylaminopeptidases,4,5 a
Pro-C-endopeptidase from Flavobacterium meningosepticum has been investigated.6
In kinetically controlled peptide synthesis, competitive
hydrolysis of the acyl enzyme and the newly formed peptide
bond are the most important yield-limiting factors. Freezing
Address reprint requests to Dr. M. Haensler, Leipzig University, Institute
of Biochemistry, Talstrasse 33, D-04103, Leipzig, Germany
Received 23 July 1997; revised 18 November 1997; accepted 4 December
1997
Enzyme and Microbial Technology 22:617– 620, 1998
© 1998 Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
the reaction mixture has been developed as an approach to
suppress these undesired side reactions.7
In this study, the capability of recombinant prolinespecific proteinase Pro-C from F. meningosepticum to form
Pro-X bonds in frozen aqueous systems was investigated for
the first time.
Materials and methods
Reagents
Amino acids, dipeptides, and derivatives thereof were obtained
from Bachem (Bubendorf, Switzerland). Recombinant endoproteinase Pro-C from E. coli, originally produced in F. meningosepticum (charge number 45167; 3.25 U ml21 determined photometrically, Z-Gly-Pro-pNA as substrate) was a gift from Fluka (Buchs,
Switzerland). Acetonitrile (HPLC grade) and trifluoroacetic acid
were from Merck (Darmstadt, Germany). Bz-Gly-Pro-OMe was
synthesized from Bz-Gly-OH and H-Pro-OMe using the mixed
anhydride method. The product was characterized by 1H-NMR.
CHN analysis was within acceptable limits.
Enzyme-catalyzed peptide synthesis
At 25°C, peptide synthesis experiments were performed in
polypropylene tubes in a total volume of 1 ml. Eight identical
samples of 0.1 ml were prepared for each peptide synthesis
experiment at 23°C. Solutions of the acyl donor ester and the
nucleophilic amino component in water were adjusted to the
desired pH. Endoproteinase Pro-C, dissolved in 0.05 m phosphate
buffer pH 7 and stored at 218°C, was added to give a final enzyme
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concentration of 0.027 mm (25°C) and 0.27 mm (23°C), respectively. Samples were stirred at 25°C or, if provided for freezing,
treated in the following manner. Prior to enzyme addition, reagents
solutions were cooled to 0°C. Enzyme solutions were added and
samples rapidly shaken and shock frozen in liquid nitrogen for
20 s. After shock freezing, samples were transferred into a cryostat
(Haake, Karlsruhe, Germany) and incubated at 23°C.
After definite reaction times, 0.1 ml aliquots, taken from the
room temperature sample, and frozen 0.1 ml samples, respectively,
were stopped by addition of an equivalent volume of trifluoroacetic acid (2.5%, v/v). The ester concentration was 2 mm, and the
concentration of the nucleophilic amino components were varied
according to their pKa to give a free base concentration of 40 mm.
pKa values8 were determined titrimetrically at a concentration of
the amino component of 6.7 mm in 0.2 m NaCl.
HPLC analysis
HPLC analysis was performed using a Shimadzu LC 10A HPLC
system and a Compaq personal computer with LC-10 software. A
Lichrospher RP-18 column (5 mm, 250 3 4 mm, Merck) was used.
Isocratic elution was performed using mixtures of acetonitrile/
water containing trifluoroacetic acid (0.1%, v/v). The relative
concentrations of substrate, hydrolysis, and aminolysis product
were calculated from the peak areas detected at 254 nm. Since
substrate, hydrolysis and peptide product (except reactions with
H-Phe-NH2 and H-Tyr-NH2 as nucleophile) contain the same
chromophor, their molar absorption coefficients were assumed to
be equal. The ratio of the molar absorption coefficients of the
substrate and peptide products containing Phe and Tyr was
determined as described by Ullmann and Jakubke.9 Peak areas
were found to be linearly dependent on the peptide concentration
of the sample. Peptide products could be detected down to 0.5% of
the initial substrate concentration. All peptide yields represent the
mean value of the results of two independent experiments.
For frozen samples at 23°C, yields are given after complete
ester consumption. During the reaction time of 24 h, no secondary
hydrolysis of the newly formed peptide bond was observed. The
yields at 25°C represent maximal yields after 5 h when about 20%
of the substrate ester was still present in the reaction mixture.
Further ester consumption was accompanied by secondary hydrolysis of the aminolysis product.
Results and discussion
In order to optimize the reaction conditions, the reverse
action of proline-specific endoproteinase was studied at
various temperatures, pH, and nucleophile concentrations.
The condensation of Bz-Gly-Pro-OMe and H-Leu-NH2 was
used as a model reaction. It has been reported that various
serine and cysteine proteases catalyze peptide bond formation in frozen reaction mixtures at 215°C.7 Surprisingly at
215°C, no endoproteinase Pro-C-catalyzed consumption of
the acyl donor ester could be observed. At 25°C, the
reaction was still strongly retarded; therefore, a reaction
temperature of 23°C which guaranteed complete ester
consumption within 24 h was chosen for further experiments. During this time, the reaction mixture remained
macroscopically frozen. The pH optimum of the enzyme
was reported to be 7,10 but at this pH, the protonation state
of the a-amino group of the nucleophilic amino components
used (pKa values varying from 7.8 –9.6) does not favor
peptide synthesis. For this reason, pH was varied from
7.5– 8.5 using identical free base concentrations of H-LeuNH2. Because no significant influence of the pH on peptide
618
Enzyme Microb. Technol., 1998, vol. 22, May 15
Table 1 Endoproteinase Pro-C-catalyzed condensation of BzGly-Pro-OMe with amino acid amides
Peptide yield (%)
Amino component
H-Leu-NH2
H-Val-NH2
H-Ile-NH2
H-Ala-NH2
H-Pro-NH2
H-Phe-NH2
H-Tyr-NH2
H-Arg-NH2
H-Lys-NH2
H-Asp-NH2
H-Glu-NH2
23°C
25°C
73
23
32
20
0
0a
0a
0a
15
24
30
14
10
12
6
0
15
12
6
3
5
9
[Bz-Gly-Pro-OMe] 5 2 mM; [amino component] 5 40 mM (free
base); [enzyme] 5 0.27 mM (23°C), 0.027 mM (25°C), pH 8.5.
Reaction time: 24 h (23°C), 5 h (25°C)
a
No ester conversion
yield was observed, all further experiments were performed
at pH 8.5 for economic reasons. At this pH, the effective
concentration of H-Leu-NH2 was varied (20 – 40 mm) at
25°C as well as at 23°C. A 20-fold excess of nucleophilic
free base gave the highest peptide yield and was used in
further studies.
Starting from this optimized reaction conditions, Bz-GlyPro-OMe was coupled with a number of amino acid amides
(Table 1).
With the exception of H-Pro-NH2 which was not accepted at all, the aromatic amino acid amides and H-ArgNH2, freezing the reaction mixture resulted in significantly
higher peptide yields. The yield-enhancing effect of freezing has been attributed to the concentration of the reactants
in the unfrozen liquid microinclusions of a partially frozen
solution.11,12 When a solution is frozen in a temperature
range wherein no eutectics are formed, the concentration of
the reactants in the remaining unfrozen liquid phase will
only depend on the temperature. One has to take into
consideration that at 23°C, which is the preferred temperature for proline-specific endopeptidase, the degree of
freeze concentration will be lower than at 215°C7; therefore, a less effective suppression of acyl enzyme hydrolysis
has to be taken into account.
H-Leu-NH2 proved to be the most effective nucleophilic
amino component. Using H-Phe-NH2, H-Tyr-NH2, and
H-Arg-NH2 as amino component, no acyl donor ester
conversion was observed under frozen state conditions.
Most probably, the enzyme activity is inhibited due to the
concentration of the amino component in the unfrozen
liquid phase. To confirm this assumption, a synthesis
experiment was performed at 25°C using 15-fold concentrated solutions of the ester substrate and H-Arg-NH2. No
ester consumption was observed under these conditions.
Krieg and Wolf6 reported on a dramatical slowdown of
proline-specific endoproteinase activity in the presence of
high concentration of H-Phe-NH2 in solution.
The yield-enhancing effect of freezing was also observed
in endoproteinase Pro-C-catalyzed coupling of Bz-Gly-Pro-
Endoproteinase: M. Haensler and H.-D. Jakubke
Figure 1 Endoproteinase Pro-C-catalyzed condensation of BzGly-Pro-OMe with free amino acids and dipeptides. [Bz-Gly-ProOMe] 5 2 mM; [amino component] 5 40 mM (free base);
[enzyme] 5 0.27 mM (23°C), 0.027 mM (25°C), pH 8.5. Reaction
time: 24 h (23°C), 5 h (25°C)
OMe with a number of free amino acids and dipeptides
(Figure 1). Dipeptides were chosen in order to study the
influence of charged amino acids in the P29-Position (nomenclature according Schechter and Berger13) as well as
that of proline compared to the aliphatic amino acid alanine.
Using H-Ala-Asp-OH as amino component, the low acceptance of Asp in the P29-position could not be compensated
by freezing-induced concentration of the reactants.
In endoproteinase Pro-C-catalyzed condensation of AcTyr-Pro-OBzl and various amino acid amides at room
temperature, Krieg and Wolf6 used about a 130-fold excess
of the nucleophile as free base. The peptide yields obtained
by these authors are comparable to that we obtained under
frozen state conditions using only a 20-fold excess of amino
component (Table 1).
Because H-Leu-NH2 is an outstanding effective nucleophile under frozen state conditions, the nucleophilic efficiency of amino components containing leucine in the
P19-position was studied (Table 2). Surprisingly, H-D-LeuNH2 was well accepted in the frozen state indicating that the
high stereospecificity of the S19-subsite reported by YoshiTable 2 Endoproteinase Pro-C-catalyzed condensation of BzGly-Pro-OMe with leucine-containing amino components
Peptide yield (%)
Amino component
H-D-Leu-NH2
H-Leu-Ala-NH2
H-Leu-Leu-NH2
H-Leu-Ala-OH
H-Leu-Pro-OH
H-Leu-b-Ala-OH
23°C
25°C
56
41
48
51
38
42
2
20
16
28
21
29
[Bz-Gly-Pro-OMe] 5 2 mM; [amino component] 5 40 mM (free
base); [enzyme] 5 0.27 mM (23°C), 0.027 mM (25°C), pH 8.5.
Reaction time: 24 h (23°C), 5 h (25°C)
moto et al.10 is changed in the frozen aqueous system. In
P29-position, no difference concerning the efficiency of
H-Ala-OH and H-b-Ala-OH was observed. Generally,
amino components containing leucine in the P19-position
proved to be the most effective nucleophiles under frozen
state conditions; however, the yield-increasing effect of
freezing strongly depends on the nucleophilic amino component resulting in yield enhancement differing from only
3% (H-Ala-Asp-OH) to 59% (H-Leu-NH2). From this
different influence of freezing, it can be concluded that
freeze concentration cannot be the only cause of the
yield-increasing effect. This assumption has also been
supported by peptide synthesis experiments in highly concentrated reactant solutions at room temperature which
failed to simulate the reaction conditions in frozen systems14 and changes in specificity of proteases observed
under frozen state conditions.15,16 Probably in frozen aqueous systems, the binding of the nucleophilic amino component is changed due to an altered conformation of the
enzyme molecule. Recently, Strambini and Gabillieri17
reported on partial freezing-induced unfolding of proteins.
Conclusions
In conclusion, endoproteinase Pro-C is a suitable catalyst in
Pro-Leu bond formation in ice; however, the scope of
applicability of the enzyme in frozen state peptide synthesis
is limited by the only moderate peptide yields obtained and
its incapability to couple aromatic amino acids and arginine
in P19-position.
Strongly amino component-depending yields obtained in
frozen systems suggest that besides the freeze concentration
effect, other factors are involved in yield enhancement by
freezing.
List of symbols
Bz,
OBzl,
HPLC
n-benzoyl-;
Benzyl ester;
High performance liquid chromatography.
Other abbreviations of amino acids, amino acid derivatives,
and peptides are according to the guidelines of the IUPACIUB Commission on Biochemical Nomenclature.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (grant number Ja 559/5-2 and INK 23/A1-1) and
the Fonds der Chemischen Industrie. We wish to thank
Degussa and E. Merck for special chemicals, Dr. Wohlgemuth, Fluka Chemie AG for endoproteinase Pro-C, Frank
Bordusa (Leipzig) for the substrate Bz-Gly-Pro-OMe, and
Mrs. Hella Spaete for skillful technical assistance.
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