Application of carboxypeptidase C for peptide synthesis D. Steinke and M. R. Kula Institut fiir E n z y m t e c h n o l o g i e , Heinrich H e i n e Universitdt Diisseldor]~ Jiilich, F R G
Carboxypeptidase C partially purified from the.flavedo sff"citrus fruit by a new, simple procedure was studied as a catalyst for peptide-bond formation. Dipeptides were obtained in high yields (80-95%) with B z - - T y r - - O E t as carboxyl-compound, and amino acid amides and amino acid alkylesters as nucleophiles. To characterize the synthesis reaction, a number o f parameters such as pH, excess o f the nucleophile, and the molarity o f the buffer were evaluated. The yield o f dipeptides depends on the side chain o f the amino acid alkylester used as the carboxyl component as well as on the N-terminul protecting group. Esterase activity was minimal in the absence o f a nucleophile, suggesting a modified mechanism for the synthesis reaction compared to other serine proteases. No secondary hydrolysis o f the peptides formed was observed.
Keywords: Carboxypeptidase C; citrus fruit: enzymatic peptide synthesis: enzyme mechanism
Introduction
Materials and methods
The formation of peptide bonds catalyzed by proteolytic enzymes has been the subject of investigations for several years, because this method offers some advantages over conventional chemical methods of synthesis. t-4 The reaction results in optically pure products 56 and takes place in aqueous media under mild conditions. Side-chain protection of trifunctional amino acids is not required. The use of serine or thioiproteases provides a fast and easy way for the synthesis of peptides without the necessity for shifting the equilibrium of the synthesis reaction by removing the product. This so-called kinetically controlled approach is based on the catalytic mechanism of serine and thiolproteases. An acyl-enzyme intermediate is formed 7,8 that can be cleaved either by water (hydrolysis) or by an amino acid or peptide derivative (aminolysis), resulting in a newly formed peptide bond. The specificity of hydrolytic enzymes presents a limiting factor for the general application of enzymatic peptide synthesis. Therefore, more enzymes need to be evaluated for this purpose. In the present paper, we report the isolation and characterization of the serine exoprotease citrus carboxypeptidase C 9 with respect to aminolysis reactions. Literature data on the influence of the amino acid side chain on peptidase activity suggested a rather broad substrate spectrum. 9
Chemicals
Address reprint requests to Professor Kula at the Institute of Enzyme Technology, Heinrich Heine Universit~t D~isseldorf, KFA Jtilich, P.O. Box 2050, 5170 Ji~lich, FRG Received 18 July 1989; revised 17 October 1989
836
Amino acid amides, N-protected amino acid esters, and the dipeptide derivative Z - - L e u - - P h e - - O H were purchased from Serva Chemicals (Heidelberg, FRG). All other chemicals employed were of analytical grade and obtained from Bachem (Bubendorf), Sigma (Deisenhofen), Merck (Darmstadt, FRG), Fluka (Buchs, FRG), or Pharmacia (Uppsala, Sweden). F o r - - T y r - - O P r o p and Z - - T y r - - O M e were prepared by Dr. Fl6rsheimer. 6 Z - - P r o - - O M e was prepared by Dr. Bernd, Degussa Wolfgang. All amino acids and amino acid derivatives had S-configuration; all values reported refer to the L-form.
Peptidase
assay of carboxypeptidase
C
Peptidase activity was determined by the ninhydrin method, J0,~ using Z - - L e u - - P h e - - O H 9 as a substrate. Citrate buffer, 800-900/zl 0.1 M, pH 5.4, 5 mM Z - L e u - - P h e - - O H , and 100-200/~1 enzyme solution (total volume I ml) were incubated at 20°C for 5-15 min. The reaction was stopped by adding 1 ml ninhydrin solution and heating immediately to 95°C. After 30 min at 95°C, the samples were cooled in ice, and 5 ml ethanol/water (! : 1) was added subsequently. The absorbance was measured at 570 nm on a Shimadzu photometer UV-160 against a blank containing no substrate. Calibration was carried out with phenylalanine. One unit of enzyme activity corresponds to 1 mol H - Phe--OH liberated in 1 min.
Enzyme Microb. Technol., 1990, vol. 12, November
© 1990 Butterworth-Heinemann
Application of carboxypeptidase C for peptide synthesis: D. Steinke and M. R. Kula
Peptide synthesis If not specified further, enzymatic reactions were carried out in 0.1 M phosphate buffer using 20 mM substrate-ester and 200 mM nucleophile (amino acid amides or esters) at 20°C in a total volume of 2 ml. If necessary, the ester was solubilized by addition of ethanol (10-40%) in the reaction mixture. The pH was adjusted by autotitration using a Radiometer Vit 90 video titrator with an ABU 91 autoburette and a TTA 80 titration assembly equipement. The reaction was started by addition of 150/~l purified enzyme solution (fraction F5, Table 1). Aliquots of 100/~1 were taken after 5, 15, 30 . . . min and mixed with the same amount of acetic acid in order to stop the reaction. The amount of dipeptide formed was determined by HPLC analysis and the integration of the corresponding peaks. One unit of synthesis activity corresponds to I /~mol dipeptide formed in 1 min. Esterase activity corresponds to l /~mol of ester substrate converted in I min. The ratio of dipeptide formed to the substrate ester converted is called selectivity. Selectivity is identical with dipeptide yield at 100% conversion of the substrate ester. The term "nucleophile" generally refers to the amino acid amides or esters.
Determination of protein The protein concentration was determined by the method of Bradford 12 using bovine serum albumin for calibration.
Analytical methods For product control as well as determination of enzyme activity and selectivities, HPLC analysis was carried out using Gyncotek equipment (Germering, FRG) with an ODS-Hypersil (5 /xm) 4.6 × 250-mm HPLC column at a flow rate of I ml/min using 60% 10 mM TBA (A)/40% acetonitrile (B). Samples were detected at 280 nm. Amino acid analysis was carried out with an Amino Acid Analyzer LC 5001 (Biotronik;
400-
T
20-
0.6
iV
:
froction siz.: 5 ml
1^.5
o
E
Puchheim, FRG). The isoelectric point and purity of the enzyme was determined with a Pharmacia Phast system using a gel in the pH range from 3 to 10.
Identification of the peptides If no reference substances were available, the products of enzymatic peptide synthesis were separated by repeated thin-layer chromatography on Merck TLC plates silica gel 60 F254 [solvent system: (I) acetic acid/ n-butanol/water (1:3: 1); (II) methanol/acetic acid/ water (60:35:5)]. The compounds visualized at 254 nm were scraped off the plate and eluted with propanol/water (1 : 1). After drying in a vacuum centrifuge (Univapo UVC 150 H), hydrolysis was carried out in 6 N HCI at I I0°C for 12 h under nitrogen. The substances were dried again for amino acid analysis.
Results and discussion
Preparation of the enzyme The flavedo of 10 oranges obtained from the local market was scraped off the fruit with a rasp and homogenized in 1 1 of 2.3% NaCl in order to solubilize the proteins. The extract was cleared by centrifugation with a Sorvall RC 5B centrifuge (10.000 rpm, 20 min) and filtration. The enzyme was isolated by fractionation with ammonium sulfate. The 30% pellet was found to contain no activity and was discarded. The pellet at 70% saturation was diluted in 0.1 M citrate buffer (pH 5.4) to a volume of 100 ml and desaited by Sephadex G-10 gel filtration using the same buffer for elution. The void fraction of the gel filtration column was concentrated by ultrafiltration using an Amicon CEC1 (Witten, FRG) equipped with a YM 10,000 membrane. The enzyme could be purified by ion-exchange chromatography on CM-Sephadex C-50 as shown in Figure 1. The enzyme appeared to be nearly pure as shown in IEF. The isoelectric point was pH 4.4, which is in agreement with the literature data for CPD-C from orange peels.13 The purification is summarized in Table 1; peptidase activity was used to monitor CPD-C activity during preparation. The enzyme concentrated by ultrafiltration was made 50% in glycerol and stored at -20°C. Using the same protocol, CPD-C was also isolated from the flavedo of citrus fruits; the final preparation had a specific activity of 0.8 U/mg.
.~ 200
Peptide synthesis .~
100
0.5
10
20
30
40
50
60
n u m b e r of f r o c t i o n
Figure I Purification of CPD-C on a CM-Sephadex C-50 column (2.6 x 35 cm). Volume of the sample was 5 ml, fraction size was 5.5 ml. The protein was eluted with a 0.1-0.3 M gradient of citrate buffer (pH 5.5) at a flow rate of 0.8 ml/min. The effluent was monitored at 280 nm
The enzyme was previously shown to have a broad specifity, considering the hydrolytic reaction, but to exhibit a preference for aromatic amino acids at the Cterminal end of proteins. 9 Therefore, B z - - T y r - - O E t was chosen as the ester compound. To characterize the synthesis reaction, B z - - T y r - OEt and H - - L e u - - N H 2 were used as standard substrates. The formation of B z - - T y r - - L e u - - N H 2 , due to the aminolysis reaction, and B z - - T y r - - O H , resulting from the hydrolysis of the acyl-enzyme intermedi-
Enzyme Microb. Technol., 1990, vol. 12, November
837
Papers Table 1
Purification of CPD-C from the flavedo of orange fruits Volume (ml)
Extract from 500 g flavedo (F1) 30-70% fraction (F2) G-10 fraction (F3) Ion-exchange c h r o m a t o g r a p h y (F4) Fraction after ultrafiltration (F5)
Activity (mU/ml)
625 185 138 100 45
Protein (mg/ml)
26
0.15 0.44 0.42 0.06 0.12
96 98 191
ate (Figure 2), was followed first as a function of pH. Corresponding to the increase of deprotonized c~amino groups, the amount of B z - - T y r - - L e u - - N H 2 increases with pH up to a value around 8.6 [Figure 3 ((3-)]. The decrease in the concentration of dipeptide above pH 9.0 can be attributed mainly to an incomplete conversion of the ester substrate due to the instability of the enzyme in this pH range [Figure 3 (-II-)]. The duration of the reaction can be attributed to a low concentration of enzyme (18/zg/ml reaction volume). No decrease in selectivity was detected in this pH range. The optimum pH with regard to peptide bond formation was therefore considered to be pH 8.5-8.6.
80 -
o
60-
5
°
I
0.--It
&: CO
• synthesis octivity
................. 6.5 7.0 7.5
6.0
8.0
i ........ 8.5 9.0
i .... T'' 9.5 10.0
0.5
pH Figure 3 Effect of different pH values on the dipeptide yield (©-), synthesis activity (-I-), and ester conversion (-II-) of the CPD-C-catalyzed formation of B z - - T y r - - L e u - - N H 2 . Reaction condition: 0.1 M phosphate buffer (pH 6.0-8.5) or 0.1 M carbonate buffer (pH 8.5-9.5); 22 mM estersubstrate; 200 mM H - - L e u - NH2; 40% ethanol; 19/~g of enzyme; total v o l u m e 2 ml
100
Bz-Tyr-Leu-NH
/
o
60-
"~
40 ~ rnl~
60-
4o-
:J
•
~ ester converted
~o
.
~ selectivity
2O
20-
concantrotion of 8z-Tyr-OEt: 22 mM
..~
A
o 80
react;on time
100
120
140
t [rain]
0 /! . . . . . . . . 0
, .........
200
~. . . . . . . . .
400
, ....
600
concentration of the nucleophile [raM]
60
Figure 2 Synthesis of B z - - T y r - - L e u - - N H 2 using CPD-C. Reaction condition: 17 mM B z - - T y r - - O E t ; 140 mM H - - L e u - - N H 2 in 0.1 M phosphate buffer adjusted to pH 8.5 with 1 M NaOH; 33% ethanol, 19/~g of enzyme; total v o l u m e 2 ml. Precipitation of the dipeptide after 30 min
838
.2
Bz-Tyr-Leu-NH:
E u
60
'~
40-
¢
40
40
/y" g ester convertea
J "
20
~8
¢}
/ I
20. / / / _
13.2 9.8 8.5
D
~>3
Z~
NI ~ -~,l -N
229 1633 1591
o~-'-°
100
60-
16.0
2z
100-
~0
170
,/---,--~
100
~--
The selectivity of the reaction increased with an increasing concentration of nucleophile ( H - - L e u - NH2), but did not come up to 100% (Figure 4). The pronounced specificity for amino acid amides as well as the lack of ester conversion in the absence of nucleophile presents further evidence for a binding site of the enzyme for the nucleophile. The existence of binding sites has been discussed including overlapping I-~ (SI) or nonoverlapping binding sites j4 for the nucleophile and the leaving group as well as conformationai changes. 15 CPD-C shows significant deviations
L®I
Total units (U)
from these models since the amount of ester converted at pH 8.5 depends on the concentration of nucleophile (Figure 4). The nucleophile seems to be essential for both, the aminolysis and hydrolysis of the ester substrate, indicating a binding of nucleophile to either the
Influence of the nucleophile
80-
Specific activity (mU/mg)
Figure 4 Influence of the amount of nucleophile on selectivity (-A-) and amount of ester converted (-&-). Reaction conditions were 0.1 M carbonate buffer, pH 8.5; ester concentration 22 mM; nucleophile concentration between 22.8 and 650 raM; 19 /~g of enzyme; total v o l u m e 2 ml. The concentration of nucleophile, where the selectivity is half-maximal, was calculated to be 87.6 mM
E n z y m e M i c r o b . T e c h n o l . , 1990, vol. 12, N o v e m b e r
Application of carboxypeptidase C for peptide synthesis: D. Steinke and M. R. Kula free enzyme or the acylated enzyme prior to deacylation (Scheme 1). The enzyme was shown to be quite specific for the side chain of the amino acid amide added (Table 2). This also supports an intermediate binding of the nucleophile. A conversion of the ester could not be detected by using H - - P r o - - N H 2 or H - - G I y - - N H 2 as nucleophiles. In the presence of the 10 mM, H - - P r o - NH2 formation of B z - - T y r - - L e u - - N H 2 was markedly inhibited, yielding only 40% as compared to 85% of dipeptide formed in the absence of H - - P r o - - N H 2 (Table 2). An aminolysis with H - - A r g - - N H 2 , H - Lys--NH2, H - - A I a - - N H 2 , and H - - L e u - - N H 2 resulted in high yields; H - - A r g - - O M e as nucleophile was less efficient.
Specificity in P1 (ester compound) Various N-protected amino acid esters were investigated as ester compounds in the synthesis reaction.
Table 2 pound
Specificity of CPD-C concerning the nucleophile com-
Nucleophile
Aminolysis product
Yield (%)
H--Leu--NH2 H--Arg--NH2 H--Ala--NH2 H--Tyr--NH2 H--His--NH2 H--VaI--NH2 H--Gly--NH2 H--Pro--NH2 H--Arg--OEt H--Arg--OH H--Lys--OH
Bz--Tyr--Leu--NH2 Bz--Tyr--Arg--NH2 Bz--Tyr--Ala--NH~ Bz--Tyr--Tyr--NH2 Bz--Tyr--His--NH2 Bz--Tyr--VaI--NH2 Bz--Tyr--Gly--NH2 Bz--Tyr--Pro--NH2 Bz--Tyr--Arg--OEt Bz--Tyr--Arg--OH Bz--Tyr--Lys--OH
85 79 74 5 0 0 0 0 17
Carboxypeptidase C partially purified from the.flavedo sff"citrus fruit by a new, simple procedure was studied as a catalyst for peptide-bond formation. Dipeptides were obtained in high yields (80-95%) with B z - - T y r - - O E t as carboxyl-compound, and amino acid amides and amino acid alkylesters as nucleophiles. To characterize the synthesis reaction, a number o f parameters such as pH, excess o f the nucleophile, and the molarity o f the buffer were evaluated. The yield o f dipeptides depends on the side chain o f the amino acid alkylester used as the carboxyl component as well as on the N-terminul protecting group. Esterase activity was minimal in the absence o f a nucleophile, suggesting a modified mechanism for the synthesis reaction compared to other serine proteases. No secondary hydrolysis o f the peptides formed was observed.
Keywords: Carboxypeptidase C; citrus fruit: enzymatic peptide synthesis: enzyme mechanism
Introduction
Materials and methods
The formation of peptide bonds catalyzed by proteolytic enzymes has been the subject of investigations for several years, because this method offers some advantages over conventional chemical methods of synthesis. t-4 The reaction results in optically pure products 56 and takes place in aqueous media under mild conditions. Side-chain protection of trifunctional amino acids is not required. The use of serine or thioiproteases provides a fast and easy way for the synthesis of peptides without the necessity for shifting the equilibrium of the synthesis reaction by removing the product. This so-called kinetically controlled approach is based on the catalytic mechanism of serine and thiolproteases. An acyl-enzyme intermediate is formed 7,8 that can be cleaved either by water (hydrolysis) or by an amino acid or peptide derivative (aminolysis), resulting in a newly formed peptide bond. The specificity of hydrolytic enzymes presents a limiting factor for the general application of enzymatic peptide synthesis. Therefore, more enzymes need to be evaluated for this purpose. In the present paper, we report the isolation and characterization of the serine exoprotease citrus carboxypeptidase C 9 with respect to aminolysis reactions. Literature data on the influence of the amino acid side chain on peptidase activity suggested a rather broad substrate spectrum. 9
Chemicals
Address reprint requests to Professor Kula at the Institute of Enzyme Technology, Heinrich Heine Universit~t D~isseldorf, KFA Jtilich, P.O. Box 2050, 5170 Ji~lich, FRG Received 18 July 1989; revised 17 October 1989
836
Amino acid amides, N-protected amino acid esters, and the dipeptide derivative Z - - L e u - - P h e - - O H were purchased from Serva Chemicals (Heidelberg, FRG). All other chemicals employed were of analytical grade and obtained from Bachem (Bubendorf), Sigma (Deisenhofen), Merck (Darmstadt, FRG), Fluka (Buchs, FRG), or Pharmacia (Uppsala, Sweden). F o r - - T y r - - O P r o p and Z - - T y r - - O M e were prepared by Dr. Fl6rsheimer. 6 Z - - P r o - - O M e was prepared by Dr. Bernd, Degussa Wolfgang. All amino acids and amino acid derivatives had S-configuration; all values reported refer to the L-form.
Peptidase
assay of carboxypeptidase
C
Peptidase activity was determined by the ninhydrin method, J0,~ using Z - - L e u - - P h e - - O H 9 as a substrate. Citrate buffer, 800-900/zl 0.1 M, pH 5.4, 5 mM Z - L e u - - P h e - - O H , and 100-200/~1 enzyme solution (total volume I ml) were incubated at 20°C for 5-15 min. The reaction was stopped by adding 1 ml ninhydrin solution and heating immediately to 95°C. After 30 min at 95°C, the samples were cooled in ice, and 5 ml ethanol/water (! : 1) was added subsequently. The absorbance was measured at 570 nm on a Shimadzu photometer UV-160 against a blank containing no substrate. Calibration was carried out with phenylalanine. One unit of enzyme activity corresponds to 1 mol H - Phe--OH liberated in 1 min.
Enzyme Microb. Technol., 1990, vol. 12, November
© 1990 Butterworth-Heinemann
Application of carboxypeptidase C for peptide synthesis: D. Steinke and M. R. Kula
Peptide synthesis If not specified further, enzymatic reactions were carried out in 0.1 M phosphate buffer using 20 mM substrate-ester and 200 mM nucleophile (amino acid amides or esters) at 20°C in a total volume of 2 ml. If necessary, the ester was solubilized by addition of ethanol (10-40%) in the reaction mixture. The pH was adjusted by autotitration using a Radiometer Vit 90 video titrator with an ABU 91 autoburette and a TTA 80 titration assembly equipement. The reaction was started by addition of 150/~l purified enzyme solution (fraction F5, Table 1). Aliquots of 100/~1 were taken after 5, 15, 30 . . . min and mixed with the same amount of acetic acid in order to stop the reaction. The amount of dipeptide formed was determined by HPLC analysis and the integration of the corresponding peaks. One unit of synthesis activity corresponds to I /~mol dipeptide formed in 1 min. Esterase activity corresponds to l /~mol of ester substrate converted in I min. The ratio of dipeptide formed to the substrate ester converted is called selectivity. Selectivity is identical with dipeptide yield at 100% conversion of the substrate ester. The term "nucleophile" generally refers to the amino acid amides or esters.
Determination of protein The protein concentration was determined by the method of Bradford 12 using bovine serum albumin for calibration.
Analytical methods For product control as well as determination of enzyme activity and selectivities, HPLC analysis was carried out using Gyncotek equipment (Germering, FRG) with an ODS-Hypersil (5 /xm) 4.6 × 250-mm HPLC column at a flow rate of I ml/min using 60% 10 mM TBA (A)/40% acetonitrile (B). Samples were detected at 280 nm. Amino acid analysis was carried out with an Amino Acid Analyzer LC 5001 (Biotronik;
400-
T
20-
0.6
iV
:
froction siz.: 5 ml
1^.5
o
E
Puchheim, FRG). The isoelectric point and purity of the enzyme was determined with a Pharmacia Phast system using a gel in the pH range from 3 to 10.
Identification of the peptides If no reference substances were available, the products of enzymatic peptide synthesis were separated by repeated thin-layer chromatography on Merck TLC plates silica gel 60 F254 [solvent system: (I) acetic acid/ n-butanol/water (1:3: 1); (II) methanol/acetic acid/ water (60:35:5)]. The compounds visualized at 254 nm were scraped off the plate and eluted with propanol/water (1 : 1). After drying in a vacuum centrifuge (Univapo UVC 150 H), hydrolysis was carried out in 6 N HCI at I I0°C for 12 h under nitrogen. The substances were dried again for amino acid analysis.
Results and discussion
Preparation of the enzyme The flavedo of 10 oranges obtained from the local market was scraped off the fruit with a rasp and homogenized in 1 1 of 2.3% NaCl in order to solubilize the proteins. The extract was cleared by centrifugation with a Sorvall RC 5B centrifuge (10.000 rpm, 20 min) and filtration. The enzyme was isolated by fractionation with ammonium sulfate. The 30% pellet was found to contain no activity and was discarded. The pellet at 70% saturation was diluted in 0.1 M citrate buffer (pH 5.4) to a volume of 100 ml and desaited by Sephadex G-10 gel filtration using the same buffer for elution. The void fraction of the gel filtration column was concentrated by ultrafiltration using an Amicon CEC1 (Witten, FRG) equipped with a YM 10,000 membrane. The enzyme could be purified by ion-exchange chromatography on CM-Sephadex C-50 as shown in Figure 1. The enzyme appeared to be nearly pure as shown in IEF. The isoelectric point was pH 4.4, which is in agreement with the literature data for CPD-C from orange peels.13 The purification is summarized in Table 1; peptidase activity was used to monitor CPD-C activity during preparation. The enzyme concentrated by ultrafiltration was made 50% in glycerol and stored at -20°C. Using the same protocol, CPD-C was also isolated from the flavedo of citrus fruits; the final preparation had a specific activity of 0.8 U/mg.
.~ 200
Peptide synthesis .~
100
0.5
10
20
30
40
50
60
n u m b e r of f r o c t i o n
Figure I Purification of CPD-C on a CM-Sephadex C-50 column (2.6 x 35 cm). Volume of the sample was 5 ml, fraction size was 5.5 ml. The protein was eluted with a 0.1-0.3 M gradient of citrate buffer (pH 5.5) at a flow rate of 0.8 ml/min. The effluent was monitored at 280 nm
The enzyme was previously shown to have a broad specifity, considering the hydrolytic reaction, but to exhibit a preference for aromatic amino acids at the Cterminal end of proteins. 9 Therefore, B z - - T y r - - O E t was chosen as the ester compound. To characterize the synthesis reaction, B z - - T y r - OEt and H - - L e u - - N H 2 were used as standard substrates. The formation of B z - - T y r - - L e u - - N H 2 , due to the aminolysis reaction, and B z - - T y r - - O H , resulting from the hydrolysis of the acyl-enzyme intermedi-
Enzyme Microb. Technol., 1990, vol. 12, November
837
Papers Table 1
Purification of CPD-C from the flavedo of orange fruits Volume (ml)
Extract from 500 g flavedo (F1) 30-70% fraction (F2) G-10 fraction (F3) Ion-exchange c h r o m a t o g r a p h y (F4) Fraction after ultrafiltration (F5)
Activity (mU/ml)
625 185 138 100 45
Protein (mg/ml)
26
0.15 0.44 0.42 0.06 0.12
96 98 191
ate (Figure 2), was followed first as a function of pH. Corresponding to the increase of deprotonized c~amino groups, the amount of B z - - T y r - - L e u - - N H 2 increases with pH up to a value around 8.6 [Figure 3 ((3-)]. The decrease in the concentration of dipeptide above pH 9.0 can be attributed mainly to an incomplete conversion of the ester substrate due to the instability of the enzyme in this pH range [Figure 3 (-II-)]. The duration of the reaction can be attributed to a low concentration of enzyme (18/zg/ml reaction volume). No decrease in selectivity was detected in this pH range. The optimum pH with regard to peptide bond formation was therefore considered to be pH 8.5-8.6.
80 -
o
60-
5
°
I
0.--It
&: CO
• synthesis octivity
................. 6.5 7.0 7.5
6.0
8.0
i ........ 8.5 9.0
i .... T'' 9.5 10.0
0.5
pH Figure 3 Effect of different pH values on the dipeptide yield (©-), synthesis activity (-I-), and ester conversion (-II-) of the CPD-C-catalyzed formation of B z - - T y r - - L e u - - N H 2 . Reaction condition: 0.1 M phosphate buffer (pH 6.0-8.5) or 0.1 M carbonate buffer (pH 8.5-9.5); 22 mM estersubstrate; 200 mM H - - L e u - NH2; 40% ethanol; 19/~g of enzyme; total v o l u m e 2 ml
100
Bz-Tyr-Leu-NH
/
o
60-
"~
40 ~ rnl~
60-
4o-
:J
•
~ ester converted
~o
.
~ selectivity
2O
20-
concantrotion of 8z-Tyr-OEt: 22 mM
..~
A
o 80
react;on time
100
120
140
t [rain]
0 /! . . . . . . . . 0
, .........
200
~. . . . . . . . .
400
, ....
600
concentration of the nucleophile [raM]
60
Figure 2 Synthesis of B z - - T y r - - L e u - - N H 2 using CPD-C. Reaction condition: 17 mM B z - - T y r - - O E t ; 140 mM H - - L e u - - N H 2 in 0.1 M phosphate buffer adjusted to pH 8.5 with 1 M NaOH; 33% ethanol, 19/~g of enzyme; total v o l u m e 2 ml. Precipitation of the dipeptide after 30 min
838
.2
Bz-Tyr-Leu-NH:
E u
60
'~
40-
¢
40
40
/y" g ester convertea
J "
20
~8
¢}
/ I
20. / / / _
13.2 9.8 8.5
D
~>3
Z~
NI ~ -~,l -N
229 1633 1591
o~-'-°
100
60-
16.0
2z
100-
~0
170
,/---,--~
100
~--
The selectivity of the reaction increased with an increasing concentration of nucleophile ( H - - L e u - NH2), but did not come up to 100% (Figure 4). The pronounced specificity for amino acid amides as well as the lack of ester conversion in the absence of nucleophile presents further evidence for a binding site of the enzyme for the nucleophile. The existence of binding sites has been discussed including overlapping I-~ (SI) or nonoverlapping binding sites j4 for the nucleophile and the leaving group as well as conformationai changes. 15 CPD-C shows significant deviations
L®I
Total units (U)
from these models since the amount of ester converted at pH 8.5 depends on the concentration of nucleophile (Figure 4). The nucleophile seems to be essential for both, the aminolysis and hydrolysis of the ester substrate, indicating a binding of nucleophile to either the
Influence of the nucleophile
80-
Specific activity (mU/mg)
Figure 4 Influence of the amount of nucleophile on selectivity (-A-) and amount of ester converted (-&-). Reaction conditions were 0.1 M carbonate buffer, pH 8.5; ester concentration 22 mM; nucleophile concentration between 22.8 and 650 raM; 19 /~g of enzyme; total v o l u m e 2 ml. The concentration of nucleophile, where the selectivity is half-maximal, was calculated to be 87.6 mM
E n z y m e M i c r o b . T e c h n o l . , 1990, vol. 12, N o v e m b e r
Application of carboxypeptidase C for peptide synthesis: D. Steinke and M. R. Kula free enzyme or the acylated enzyme prior to deacylation (Scheme 1). The enzyme was shown to be quite specific for the side chain of the amino acid amide added (Table 2). This also supports an intermediate binding of the nucleophile. A conversion of the ester could not be detected by using H - - P r o - - N H 2 or H - - G I y - - N H 2 as nucleophiles. In the presence of the 10 mM, H - - P r o - NH2 formation of B z - - T y r - - L e u - - N H 2 was markedly inhibited, yielding only 40% as compared to 85% of dipeptide formed in the absence of H - - P r o - - N H 2 (Table 2). An aminolysis with H - - A r g - - N H 2 , H - Lys--NH2, H - - A I a - - N H 2 , and H - - L e u - - N H 2 resulted in high yields; H - - A r g - - O M e as nucleophile was less efficient.
Specificity in P1 (ester compound) Various N-protected amino acid esters were investigated as ester compounds in the synthesis reaction.
Table 2 pound
Specificity of CPD-C concerning the nucleophile com-
Nucleophile
Aminolysis product
Yield (%)
H--Leu--NH2 H--Arg--NH2 H--Ala--NH2 H--Tyr--NH2 H--His--NH2 H--VaI--NH2 H--Gly--NH2 H--Pro--NH2 H--Arg--OEt H--Arg--OH H--Lys--OH
Bz--Tyr--Leu--NH2 Bz--Tyr--Arg--NH2 Bz--Tyr--Ala--NH~ Bz--Tyr--Tyr--NH2 Bz--Tyr--His--NH2 Bz--Tyr--VaI--NH2 Bz--Tyr--Gly--NH2 Bz--Tyr--Pro--NH2 Bz--Tyr--Arg--OEt Bz--Tyr--Arg--OH Bz--Tyr--Lys--OH
85 79 74 5 0 0 0 0 17
