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Isolation, Purification, and Characterization of a New Enzyme from Pseudomonas sp. M-27, Carboxypeptidase G3 a
a
a
Noriko Yasuda , Michie Kaneko & Yukio Kimura a
Faculty of Pharmaceutical Sciences, Mukogawa Women’s University, 11–68, Koshien Kyuban-cho, Nishinomiya 663, Japan Published online: 12 Jun 2014.
To cite this article: Noriko Yasuda, Michie Kaneko & Yukio Kimura (1992) Isolation, Purification, and Characterization of a New Enzyme from Pseudomonas sp. M-27, Carboxypeptidase G3, Bioscience, Biotechnology, and Biochemistry, 56:10, 1536-1540, DOI: 10.1271/bbb.56.1536 To link to this article: http://dx.doi.org/10.1271/bbb.56.1536
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (10), 1536-1540, 1992
Isolation, Purification, and Characterization of a New Enzyme from Pseudomonas sp. M-27, Carboxypeptidase G 3 Noriko YASUDA, Michie KANEKO, and Yukio KIMURA *
Downloaded by [McMaster University] at 10:18 17 December 2014
Faculty of Pharmaceutical Sciences, Mukogawa Women's University, 11-68, Koshien Kyuban-cho, Nishinomiya 663, Japan Received January 16, 1992
A new type of carboxypeptidase was found in a strain of Pseudomonas sp. M-27 isolated from soil. The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity. This enzyme had a single peak with a molecular weight of 60,000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (Mr: 15,000). The enzyme hydrolyzed predominately acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini. This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators. The enzyme resembles carboxypeptidase G 1 or G 2 in its glutamate-releasing activity. However, it acts not only on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, as this enzyme differs from carboxypeptidase G 1 or G 2 , it was named carboxypeptidase G 3 •
We found the enzyme polymyxin acylase in Pseudomonas Sp.,1) which hydrolyzes only the fatty acyl group of N-acyl-peptides without affecting the peptide moiety, and purified it to a homogeneous state. 2 ) During its purification from a polymyxin acylase-producing strain, M-27, we found another enzyme that hydrolyzed only N-fatty acyl-Glu or -Asp, but not polymyxins. This enzyme seemed to be a kind of carboxypeptidase, because it acted on carboxyl-terminal amino acids in peptides such as N-octanoyl-GlY-L-Glu or N-benzyloxycarbonyl (Z)-L-Tyr-L-Glu. Also although its optical specificity was not clear, it acted not only on the L-form but also on the D-form of acidic amino acids and had an affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, it shows some similarity to but is markedly different from the known enzyme carboxypeptidases, G 13 ) and G 2 . 4 )
Materials and Methods Materials. N-Fatty acyl amino acids were prepared by the watersoluble active ester method from the appropriate fatty acyl ester of p-hydroxyphenyldimethylsulfonium methylsulfate and amino acids. N-Acyl-peptides, insulin B chain, and N-octanoyl-p-nitroanilide were prepared in our laboratory. Several N-Z-peptides were the generous gifts of Dr. M. Fujino, Takeda Chemical Industries, Ltd. (Osaka, Japan). Colistin sulfate was kindly supplied by Banyu Pharmaceutical Co. (Tokyo, Japan). Octa-ethyleneglycol mono n-dodecyl ether (C 12 E s) was purchased from Nikko Chemicals Co., Ltd. (Tokyo, Japan). Polyglutamic acid (mean M r = 5000) and polyaspartic acid (meanMr = 5000) were obtained from Sigma Chemical Co. (U.S.A.). All other chemicals were purchased from Nacarai Tesque, Inc. (Kyoto, Japan). Enzyme assay. Carboxypeptidase or acyl amino acid acylase activities were assayed from the ninhydrin color spot of liberated amino acid on the thin layer chromatography (TLC) plate (Method 1) as follows: the reaction mixture, 200,u1, containing 10 ,umol of acyl amino acid sodium salt, I mM ZnCl 2 and the enzyme solution (20,u1) in 50 mM Tris buffer (pH 8.0), was incubated at 37°C for several hours. The amounts of liberated
*
amino acid in 100,u1 of the mixture was measured colorimetrically by the ninhydrin method. During enzyme purification and for enzyme characterization, this assay method using N-octanoyl-DL-glutamic acid was used. In the case of peptides, a l-,ul portion of the reaction mixture was developed by TLC on a silica gel plate [solvent system: upper layer of n-BuOH-AcOH-H 2 0 (4: 1: 3) containing 1/20 pyridine by volume, or phenol-H 2 0 (4: 1)]. The amount of amino acid liberated was measured by the ninhydrin method with Chromoscan 200/201 (Joyce-Loebl, England). One unit of the enzyme was defined as the amount of enzyme that catalyzed the formation of 1,umol of glutamic acid from Noctanoyl-DL-glutamic acid per min. Activities for high molecular weight peptides were measured using gel permeation chromatography (GPC) (Method 2) as follows: the activities for polyglutamic acid and polyaspartic acid were assayed using a GPC column by UV absorption of the liberated amino acids. The reaction mixture, 200,u1, containing 5,umol of peptides and the enzyme solution (50,ul) in 50 mM Tris buffer (pH 8.0), was incubated at 37°C for 24 hr. The mixture was filtered using a 0.45-,um filter (Tosoh Corp., Tokyo, Japan). A lO-,u1 portion of the filtrate was injected into the HPLC system [column, TSK gel G2000 SW (7.5 x 600mm) (Tosoh Corp., Tokyo, Japan); mobile phase, 10 mM phosphate buffer (pH 7.2) containing 0.2 MNaCI; detector, UV (210nm)]. The amount of amino acid liberated was calculated from the height of the peak on the chromatogram. Protein measurement. Protein was measured by a protein assay using bicinchoninic acid (Pierce Chemical Co., Rockford, U.S.A.) with bovine serum albumin as the standard. In all the chromatographic procedures, the protein was measured from the absorbance at 280 nm. Purification of carboxypeptidase G 3 • The enzyme was purified from an acetone-dried cell powder of Pseudomonas sp. M-27. All the buffer solutions used contained 200mM KCI and 0.2% C 12 E s, unless otherwise noted. Step 1. One gram of acetone-dried cell powder of Pseudomonas sp. M-27 was suspended in 30 ml of 20 mM phosphate buffer (pH 8.0) containing 200 mM KCI and 6 mg of colistin sulfate. The mixture was stirred at 20°C for 30 min and then centrifuged at 25,000 x g for 30 min. Step 2. The supernatant was introduced into a DEAE-cellulose (DE-53; Whatman Biosystem Ltd., U.K.) column (1.6 x 30cm) equilibrated with 20 mM phosphate buffer (pH 8.0). The enzyme was eluted with the same buffer, and then with a linear gradient from 200 to 500mM KClin the
To whom correspondence should be addressed.
Abbreviations: DFP, diisopropylphosphoro fluoridate; EGTA, ethylene glycol bis(fJ-aminoethylether)-N,N,N',N'-tetraacetic acid; GPC, gel
permeation chromatography; PCMB, p-chloromercuribenzoate; PMSF, phenylmethane sulfonylfluoride; TLC, thin layer chromatography; Z, benzyloxycarbonyl.
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Carboxypeptidase G 3 equilibration buffer. The active fractions eluted with the first buffer were combined. Step 3. The enzyme from the above step was introduced into a hydroxyapatite (Seikagaku Kogyo Co., Ltd., Tokyo, Japan) column (1.6 x 30 cm) equilibrated with 20 mM phosphate buffer (pH 8.0). The enzyme was eluted first with the same buffer and then with a linear gradIent from 20 to 500 mM phosphate buffer (pH 8.0). The active fractions emerged in two peaks (fraction I and fraction II). Fraction II, which was eluted with 500 mM phosphate buffer and pooled, had the most enzyme activity. Step 4. The enzyme was introduced into a Butyl-Toyopearl650M (Tosoh Corp., Tokyo, Japan) column (1.6 x 30cm) equilibrated with 20mM phosphate buffer (pH 8.0) containing 60% saturated ammonium sulfate and 0.05% C l2 E s. The column was washed with the same buffer, and then the enzyme was eluted first with a linear gradient from 60 to 0% saturated ammonium sulfate in 20 mM phosphate buffer containing 0.05% C l2 E s . The fractions, which showed the activity for N-octanoyl-oL-glutamic acid but not for colistin, were combined and concentrated with a Millipore Immersible-CX Ultrafilter. Step 5. The concentrated enzyme was introduced into a Superdex 200 (Pharmacia LKB Biotechnology, Sweden) column (1.6 x 60cm) equilibrated with 200mM phosphate buffer (pH 8.0) containing 0.05% C l2 E s . The active fractions were pooled.
Table I. M-27
Purification of Carboxypeptidase G 3 from Pseudomonas sp.
Step
Total protein (mg)
Cell-free extract DEAE-cellulose Hydroxyapatite Butyl-Toyopearl Superdex 200
144.0 78.6 26.0 5.4 0.8
10
Total activity (units) 390 327 245 130 26.7
Specific activity (units/mg)
Yield (%)
2.7 4.2 9.4 24.0 33.0
100 84 63 33 6.8
6
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2
Enzyme homogeneity. The purified enzyme was dialyzed against distilled water and lyophilized. SDS-polyacrylamide gel electrophoresis was done using ExcelGel SDS, gradient 8-18 and its buffer strips (Pharmacia LKB Biotechnology, Sweden) at 600 V. Proteins were stained with Coomassie brilliant blue R-250. Isoelectric point. Isoelectric-focusing electrophoresis was done using an Ampholine PAG plate (Pharmacia LKB Biotechnology, Sweden) containing carrier ampholyte in the pH range of 3.5 to 9.5. Electrophoresis waS done at 4°C and 1500V for 1.5h in a Multiphor II electrophoresis system (Pharmacia LKB Biotechnology, Sweden). The isoelectric point of the purified enzyme was identified using an isoelectric focusing calibration kit (pH 3~ 10) against the standard. Measurement of molecular weight. The molecular weight of the purified enzyme was measured by gel filtration on a Superdex 200 column (1.6 x 60cm) by the method of Andrews,S) using a standard protein kit (Pharmacia LKB Biotechnology, Sweden). SDS-polyacrylamide gel electrophoresis was also used to measure the molecular weight of the subunit. Optical specificity of carboxypeptidase G 3 • The reaction mixture was incubated as described above for the enzyme assay, Method 1, using N-octanoyl-oL-aspartic acid as substrate. A 10-J.d portion of the filtrate was injected into the HPLC column [Chiralpak WH (0.46 x 25 cm) (Daicel Chemical Industries, Ltd., Tokyo, Japan); mobile phase, 0.25 mM CuS0 4 ; detector, UV (254 nm); temperature, 50°C]. The ratio of o-form to L-form of the reaction product was calculated from the peak area on the chromatogram, and compared with those of the authentic 0- and L-aspartic acid.
Results Purification of carboxypeptidase G2 from Pseudomonas sp. M-27 Carboxypeptidase G 3 was readily solubilized by treatment with colistin sulfate in the same way as Triton X-IOO. In this procedure, the carboxypeptidase G 3 was slightly more rapidly liberated than polymyxin acylase. The solubilized carboxypeptidase G 3 was further purified on DEAE-cellulose, hydroxyapatite, Butyl-Toyopearl, and Superdex 200 columns. The purification procedure is summarized in Table 1. N-Octanoyl-DL-glutamic acid was adopted as a substrate for the purification procedure, because this made measurement of the activity very simple. However, N-octanoyl-DL-glutamic acid is also susceptible to hydrolysis by polymyxin acylase, which is present in the crude enzyme preparation. Thus, the activity given in Table I is higher than the actual value, because the total activity
10' '----'50---6~0---7~0----'8-0 --9~0----' Elution volume (ml)
Fig. 1. Molecular Weight Estimation of Carboxypeptidase G 3 by Gel Filtration on Superdex 200. M, standards: I) ferritin (450,000); 2) aldolase (158,000); 3) transferrin (90,000); 4)
ovalbumin (45,000); G) carboxypeptidase G 3 .
includes that which should be counted for polymyxin acylase. Finally Superdex 200 column chromatography gave a pure sample without any polymyxin acylase activity (data not shown). Physico-chemical properties The molecular weight of the enzyme was estimated to be about 60,000 by the high-performance gel filtration using a Superdex 200 column (Fig. 1). SDS-polyacrylamide gel electrophoresis gave a single band at a position corresponding to an estimated molecular weight of 15,000 (Fig. 2). These results show that the enzyme consists of four subunits of identical molecular weights. The enzyme was tested by isoelectric focusing in a gradient ranging from pH 3.5 to pH 9.5. Its isoelectric point was estimated to be 7.2. The enzyme activity was optimal at pH 8.0 for N-octanoyl-DLglutamic acid (Fig. 3A) and was stable in the pH range of 7 to 9 (Fig. 3B). The optimum temperature of the enzymatic reaction at pH 8.0 was 60°C (Fig. 4A), and the enzyme was stable up to 50°C for 30 min (Fig. 4B). The observed Vmax and K m for the substrate were 33 ,umol/min/mg and 3~5 mM, respectively. Effects of inhibitors and metals Table II shows that the enzyme was inactivated (to 3% or 8%) by incubation with 1 mM EGTA or I mM 0phenanthroline at 37°C, but was not affected by incubation
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N. YASUDA, M. KANEKO, and Y. KIMURA
1538
A
100
B
~ ~
....>. >
....u ~
41
50
>
94.0
....
6 7.0
et::
~
41
4 3.0
O'-------'--------'---...L-_-L_~L-_---L...__=______:!:==::!L._J.
40
80
40
Temperature
30.0
60
80
(CO)
Fig. 4. Effects of Temperature on Activity (A) and Stability (B) of Carboxypeptidase G 3 .
20.1
(A) Temperature-activity curve in 0.1 M phosphate buffer (pH 8.0) for 30 min of incubation. (B) Thermal stability curve. The enzyme was incubated at different temperatures in 0.1 M phosphate buffer (pH 8.0) for 30 min. The activities were assayed by method 1 as described in Materials and Methods.
14.4.
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60
Table II. Effects of Chemical Reagents and Inhibitors on Carboxypeptidase G 3 Activity
Lane
2
Fig. 2. SDS-Polyacrylamide Gel Electrophoresis of the Purified Carboxypeptidase G 3 . Electrophoresis of the purified enzyme and molecular markers was done on ExcelGel SDS, gradient 8-18. Lane I, M r standards: phosphorylase b (94,000); bovine serum albumin (67,000); ovalbumin (43,000); carbonic anhydrase (30,000); soybean trypsin inhibitor (20,100); a-lactalbumin (14,400). Lane 2, the purified enzyme.
....
100
A
B
\
~
....>. >
....u
/\
~
ell
50
~
>
.... ~
41
et::
5
6
7
8
None EGTA EGTA 0- Phenanthroline o-Phenanthroline N- Bromosuccinimide PCMB Iodoacetate PMSF DFP
5
9 10 11
Concentration (mM)
Residual activity (%)
0.1
100 29
1.0 0.1
35
3
1.0
8 6
1.0 1.0 1.0 1.0
81 94 100 94
1.0
The reaction mixture, 200 pI, consisting of enzyme (50 pI) and inhibitor at the given concentration in 50 mM phosphate buffer (pH 8) was incubated at 37°C for 20 min. The residual activity was assayed by method 1 as described in Materials and Methods. Table III.
/ 0
Chemical reagent
Effects of Metal Ions on Carboxypeptidase G 3 Activity Relative activity (%)
6
7
8
9 10 11
Metal ion (1 mM)
pH
Fig. 3. Effects of pH on Activity (A) and Stability (B) of Carboxypeptidase G 3 . (A) pH-Activity curve in the following buffers: 0, 0.1 M citrate-phosphate buffer (pH 5.0-6.0); .,0.1 M phosphate buffer (pH 6.0-8.0); 6,0.1 M Tris-HCI buffer (pH 8.0-9.0); ,A., 0.1 M carbonate buffer (pH 9.0-11.0). (B) pH-Stability curve. The enzyme was treated for 24 h at 4°C in the buffers (0.1 M) described in A. The activities were assayed by method 1 as described in Materials and Methods.
without these chelators. The inactivated enzyme was effectively activated by addition of Zn 2 +, slightly activated by Cu 2 +, Mn 2 +, and Co 2 +, and not affected by Ca 2 +, Fe 2 +, or Hg 2 + (Table III). Substrate specificity To investigate the specificity of the enzyme for the carboxyl-terminal amino acid, eleven kinds of N-octanoylamino acids were used as substrates. As shown in Table IV,
None CaCl 2 MnS0 4 FeS04 CoCl 2 Cu(CH 3 COOh ZnC1 2 HgC1 2
Native enzyme
EGTA treatment
100
2.4 4.9 29 5.2 13 54 94 0.7
66 19
6 14 4 157 0
The reaction mixture, 200 pI, consisting of enzyme (50 pI) and metal ion (1 mM) in 50 mM phosphate buffer (pH 8) was incubated at 37°C for 20 min. The activity was assayed by method 1 as described in Materials and Methods. The enzyme treated by metal ion chelating agent (20mM EGTA) was also assayed as described above.
N-octanoyl-DL-glutamic acid and -DL-aspartic acid were exclusively hydrolyzed, but the other N-octanoyl-amino acids were very slightly or not hydrolyzed. Unlike polymyxin
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Carboxypeptidase G 3 Table IV. Acids
Relative Activities of Carboxypeptidase G 3 for Acyl Amino 0.05
Substrate
Relative activity (%)
E c:
Downloaded by [McMaster University] at 10:18 17 December 2014
C'l
Acetyl-(Cz)-oL-glutamic acid Butyroyl-(C4)-oL-glutamic acid Hexanoyl-(C 6)-oL-glutamic acid Octanoyl-(Cs)-oL-glutamic acid Decanoyl-(C1o)-oL-glutamic acid Dodecanoyl-(C1z)-OL-glutamic acid Tetradecaonyl-(C 14)-OL-glutamic acid Hexadecanoyl-(C 16 )-OL-glutamic acid Z-oL-glutamic acid Benzoyl-oL-glutamic acid Octanoyl-(Cs)-oL-aspartic acid Octanoyl-(Cs)-glycine Octanoyl-(Cs)-oL-alanine Octanoyl-(Cs)-oL-valine Octanoyl-(Cs)-oL-leucine Octanoyl-(Cs)-oL-serine Octanoyl-(Cs)-oL-threonine Octanoyl-(Cs)-oL-methionine Octanoyl-(Cs)-oL-phenylalanine Octanoyl-(C s)-6-aminohexanoic acid
38 42 81 100 III 50 39
The enzyme activity for octanoyl-(Cs)-oL-glutamic acid, corresponding to 33,umol/min/mg, was taken as 100%. Table V.
Substrate Specificities of Carboxypeptidase G 3 for Peptides
Substrate Octanoyl-Gly-Gly Octanoyl-Gly-Ala Octanoyl-Gly-Met Octanoyl-Gly-Leu Octanoyl-Ala-Gly Octanoyl-Gly-Gly-Gly-Gly Z-Gly-Gly Z-Gly-Phe Z-Ser-Ser-Tyr Z-Tyr-Gly-Gly Z-Asp(OBul)-Ala-Ala Z-Glu(OBul)-Ser-Thr-Leu Insulin B chain Poly-Glu (mean M r 5000) Poly-Asp (mean M r 5000) Z-Thr-Pro Z-Ser-Glu(OBul)-Lys(Boc)-Ser-Gln-Thr-Pro
iU
0.03
III
u
c:
(lJ
0,02
.0
0
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Isolation, Purification, and Characterization of a New Enzyme from Pseudomonas sp. M-27, Carboxypeptidase G3 a
a
a
Noriko Yasuda , Michie Kaneko & Yukio Kimura a
Faculty of Pharmaceutical Sciences, Mukogawa Women’s University, 11–68, Koshien Kyuban-cho, Nishinomiya 663, Japan Published online: 12 Jun 2014.
To cite this article: Noriko Yasuda, Michie Kaneko & Yukio Kimura (1992) Isolation, Purification, and Characterization of a New Enzyme from Pseudomonas sp. M-27, Carboxypeptidase G3, Bioscience, Biotechnology, and Biochemistry, 56:10, 1536-1540, DOI: 10.1271/bbb.56.1536 To link to this article: http://dx.doi.org/10.1271/bbb.56.1536
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (10), 1536-1540, 1992
Isolation, Purification, and Characterization of a New Enzyme from Pseudomonas sp. M-27, Carboxypeptidase G 3 Noriko YASUDA, Michie KANEKO, and Yukio KIMURA *
Downloaded by [McMaster University] at 10:18 17 December 2014
Faculty of Pharmaceutical Sciences, Mukogawa Women's University, 11-68, Koshien Kyuban-cho, Nishinomiya 663, Japan Received January 16, 1992
A new type of carboxypeptidase was found in a strain of Pseudomonas sp. M-27 isolated from soil. The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity. This enzyme had a single peak with a molecular weight of 60,000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (Mr: 15,000). The enzyme hydrolyzed predominately acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini. This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators. The enzyme resembles carboxypeptidase G 1 or G 2 in its glutamate-releasing activity. However, it acts not only on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, as this enzyme differs from carboxypeptidase G 1 or G 2 , it was named carboxypeptidase G 3 •
We found the enzyme polymyxin acylase in Pseudomonas Sp.,1) which hydrolyzes only the fatty acyl group of N-acyl-peptides without affecting the peptide moiety, and purified it to a homogeneous state. 2 ) During its purification from a polymyxin acylase-producing strain, M-27, we found another enzyme that hydrolyzed only N-fatty acyl-Glu or -Asp, but not polymyxins. This enzyme seemed to be a kind of carboxypeptidase, because it acted on carboxyl-terminal amino acids in peptides such as N-octanoyl-GlY-L-Glu or N-benzyloxycarbonyl (Z)-L-Tyr-L-Glu. Also although its optical specificity was not clear, it acted not only on the L-form but also on the D-form of acidic amino acids and had an affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, it shows some similarity to but is markedly different from the known enzyme carboxypeptidases, G 13 ) and G 2 . 4 )
Materials and Methods Materials. N-Fatty acyl amino acids were prepared by the watersoluble active ester method from the appropriate fatty acyl ester of p-hydroxyphenyldimethylsulfonium methylsulfate and amino acids. N-Acyl-peptides, insulin B chain, and N-octanoyl-p-nitroanilide were prepared in our laboratory. Several N-Z-peptides were the generous gifts of Dr. M. Fujino, Takeda Chemical Industries, Ltd. (Osaka, Japan). Colistin sulfate was kindly supplied by Banyu Pharmaceutical Co. (Tokyo, Japan). Octa-ethyleneglycol mono n-dodecyl ether (C 12 E s) was purchased from Nikko Chemicals Co., Ltd. (Tokyo, Japan). Polyglutamic acid (mean M r = 5000) and polyaspartic acid (meanMr = 5000) were obtained from Sigma Chemical Co. (U.S.A.). All other chemicals were purchased from Nacarai Tesque, Inc. (Kyoto, Japan). Enzyme assay. Carboxypeptidase or acyl amino acid acylase activities were assayed from the ninhydrin color spot of liberated amino acid on the thin layer chromatography (TLC) plate (Method 1) as follows: the reaction mixture, 200,u1, containing 10 ,umol of acyl amino acid sodium salt, I mM ZnCl 2 and the enzyme solution (20,u1) in 50 mM Tris buffer (pH 8.0), was incubated at 37°C for several hours. The amounts of liberated
*
amino acid in 100,u1 of the mixture was measured colorimetrically by the ninhydrin method. During enzyme purification and for enzyme characterization, this assay method using N-octanoyl-DL-glutamic acid was used. In the case of peptides, a l-,ul portion of the reaction mixture was developed by TLC on a silica gel plate [solvent system: upper layer of n-BuOH-AcOH-H 2 0 (4: 1: 3) containing 1/20 pyridine by volume, or phenol-H 2 0 (4: 1)]. The amount of amino acid liberated was measured by the ninhydrin method with Chromoscan 200/201 (Joyce-Loebl, England). One unit of the enzyme was defined as the amount of enzyme that catalyzed the formation of 1,umol of glutamic acid from Noctanoyl-DL-glutamic acid per min. Activities for high molecular weight peptides were measured using gel permeation chromatography (GPC) (Method 2) as follows: the activities for polyglutamic acid and polyaspartic acid were assayed using a GPC column by UV absorption of the liberated amino acids. The reaction mixture, 200,u1, containing 5,umol of peptides and the enzyme solution (50,ul) in 50 mM Tris buffer (pH 8.0), was incubated at 37°C for 24 hr. The mixture was filtered using a 0.45-,um filter (Tosoh Corp., Tokyo, Japan). A lO-,u1 portion of the filtrate was injected into the HPLC system [column, TSK gel G2000 SW (7.5 x 600mm) (Tosoh Corp., Tokyo, Japan); mobile phase, 10 mM phosphate buffer (pH 7.2) containing 0.2 MNaCI; detector, UV (210nm)]. The amount of amino acid liberated was calculated from the height of the peak on the chromatogram. Protein measurement. Protein was measured by a protein assay using bicinchoninic acid (Pierce Chemical Co., Rockford, U.S.A.) with bovine serum albumin as the standard. In all the chromatographic procedures, the protein was measured from the absorbance at 280 nm. Purification of carboxypeptidase G 3 • The enzyme was purified from an acetone-dried cell powder of Pseudomonas sp. M-27. All the buffer solutions used contained 200mM KCI and 0.2% C 12 E s, unless otherwise noted. Step 1. One gram of acetone-dried cell powder of Pseudomonas sp. M-27 was suspended in 30 ml of 20 mM phosphate buffer (pH 8.0) containing 200 mM KCI and 6 mg of colistin sulfate. The mixture was stirred at 20°C for 30 min and then centrifuged at 25,000 x g for 30 min. Step 2. The supernatant was introduced into a DEAE-cellulose (DE-53; Whatman Biosystem Ltd., U.K.) column (1.6 x 30cm) equilibrated with 20 mM phosphate buffer (pH 8.0). The enzyme was eluted with the same buffer, and then with a linear gradient from 200 to 500mM KClin the
To whom correspondence should be addressed.
Abbreviations: DFP, diisopropylphosphoro fluoridate; EGTA, ethylene glycol bis(fJ-aminoethylether)-N,N,N',N'-tetraacetic acid; GPC, gel
permeation chromatography; PCMB, p-chloromercuribenzoate; PMSF, phenylmethane sulfonylfluoride; TLC, thin layer chromatography; Z, benzyloxycarbonyl.
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Carboxypeptidase G 3 equilibration buffer. The active fractions eluted with the first buffer were combined. Step 3. The enzyme from the above step was introduced into a hydroxyapatite (Seikagaku Kogyo Co., Ltd., Tokyo, Japan) column (1.6 x 30 cm) equilibrated with 20 mM phosphate buffer (pH 8.0). The enzyme was eluted first with the same buffer and then with a linear gradIent from 20 to 500 mM phosphate buffer (pH 8.0). The active fractions emerged in two peaks (fraction I and fraction II). Fraction II, which was eluted with 500 mM phosphate buffer and pooled, had the most enzyme activity. Step 4. The enzyme was introduced into a Butyl-Toyopearl650M (Tosoh Corp., Tokyo, Japan) column (1.6 x 30cm) equilibrated with 20mM phosphate buffer (pH 8.0) containing 60% saturated ammonium sulfate and 0.05% C l2 E s. The column was washed with the same buffer, and then the enzyme was eluted first with a linear gradient from 60 to 0% saturated ammonium sulfate in 20 mM phosphate buffer containing 0.05% C l2 E s . The fractions, which showed the activity for N-octanoyl-oL-glutamic acid but not for colistin, were combined and concentrated with a Millipore Immersible-CX Ultrafilter. Step 5. The concentrated enzyme was introduced into a Superdex 200 (Pharmacia LKB Biotechnology, Sweden) column (1.6 x 60cm) equilibrated with 200mM phosphate buffer (pH 8.0) containing 0.05% C l2 E s . The active fractions were pooled.
Table I. M-27
Purification of Carboxypeptidase G 3 from Pseudomonas sp.
Step
Total protein (mg)
Cell-free extract DEAE-cellulose Hydroxyapatite Butyl-Toyopearl Superdex 200
144.0 78.6 26.0 5.4 0.8
10
Total activity (units) 390 327 245 130 26.7
Specific activity (units/mg)
Yield (%)
2.7 4.2 9.4 24.0 33.0
100 84 63 33 6.8
6
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Enzyme homogeneity. The purified enzyme was dialyzed against distilled water and lyophilized. SDS-polyacrylamide gel electrophoresis was done using ExcelGel SDS, gradient 8-18 and its buffer strips (Pharmacia LKB Biotechnology, Sweden) at 600 V. Proteins were stained with Coomassie brilliant blue R-250. Isoelectric point. Isoelectric-focusing electrophoresis was done using an Ampholine PAG plate (Pharmacia LKB Biotechnology, Sweden) containing carrier ampholyte in the pH range of 3.5 to 9.5. Electrophoresis waS done at 4°C and 1500V for 1.5h in a Multiphor II electrophoresis system (Pharmacia LKB Biotechnology, Sweden). The isoelectric point of the purified enzyme was identified using an isoelectric focusing calibration kit (pH 3~ 10) against the standard. Measurement of molecular weight. The molecular weight of the purified enzyme was measured by gel filtration on a Superdex 200 column (1.6 x 60cm) by the method of Andrews,S) using a standard protein kit (Pharmacia LKB Biotechnology, Sweden). SDS-polyacrylamide gel electrophoresis was also used to measure the molecular weight of the subunit. Optical specificity of carboxypeptidase G 3 • The reaction mixture was incubated as described above for the enzyme assay, Method 1, using N-octanoyl-oL-aspartic acid as substrate. A 10-J.d portion of the filtrate was injected into the HPLC column [Chiralpak WH (0.46 x 25 cm) (Daicel Chemical Industries, Ltd., Tokyo, Japan); mobile phase, 0.25 mM CuS0 4 ; detector, UV (254 nm); temperature, 50°C]. The ratio of o-form to L-form of the reaction product was calculated from the peak area on the chromatogram, and compared with those of the authentic 0- and L-aspartic acid.
Results Purification of carboxypeptidase G2 from Pseudomonas sp. M-27 Carboxypeptidase G 3 was readily solubilized by treatment with colistin sulfate in the same way as Triton X-IOO. In this procedure, the carboxypeptidase G 3 was slightly more rapidly liberated than polymyxin acylase. The solubilized carboxypeptidase G 3 was further purified on DEAE-cellulose, hydroxyapatite, Butyl-Toyopearl, and Superdex 200 columns. The purification procedure is summarized in Table 1. N-Octanoyl-DL-glutamic acid was adopted as a substrate for the purification procedure, because this made measurement of the activity very simple. However, N-octanoyl-DL-glutamic acid is also susceptible to hydrolysis by polymyxin acylase, which is present in the crude enzyme preparation. Thus, the activity given in Table I is higher than the actual value, because the total activity
10' '----'50---6~0---7~0----'8-0 --9~0----' Elution volume (ml)
Fig. 1. Molecular Weight Estimation of Carboxypeptidase G 3 by Gel Filtration on Superdex 200. M, standards: I) ferritin (450,000); 2) aldolase (158,000); 3) transferrin (90,000); 4)
ovalbumin (45,000); G) carboxypeptidase G 3 .
includes that which should be counted for polymyxin acylase. Finally Superdex 200 column chromatography gave a pure sample without any polymyxin acylase activity (data not shown). Physico-chemical properties The molecular weight of the enzyme was estimated to be about 60,000 by the high-performance gel filtration using a Superdex 200 column (Fig. 1). SDS-polyacrylamide gel electrophoresis gave a single band at a position corresponding to an estimated molecular weight of 15,000 (Fig. 2). These results show that the enzyme consists of four subunits of identical molecular weights. The enzyme was tested by isoelectric focusing in a gradient ranging from pH 3.5 to pH 9.5. Its isoelectric point was estimated to be 7.2. The enzyme activity was optimal at pH 8.0 for N-octanoyl-DLglutamic acid (Fig. 3A) and was stable in the pH range of 7 to 9 (Fig. 3B). The optimum temperature of the enzymatic reaction at pH 8.0 was 60°C (Fig. 4A), and the enzyme was stable up to 50°C for 30 min (Fig. 4B). The observed Vmax and K m for the substrate were 33 ,umol/min/mg and 3~5 mM, respectively. Effects of inhibitors and metals Table II shows that the enzyme was inactivated (to 3% or 8%) by incubation with 1 mM EGTA or I mM 0phenanthroline at 37°C, but was not affected by incubation
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A
100
B
~ ~
....>. >
....u ~
41
50
>
94.0
....
6 7.0
et::
~
41
4 3.0
O'-------'--------'---...L-_-L_~L-_---L...__=______:!:==::!L._J.
40
80
40
Temperature
30.0
60
80
(CO)
Fig. 4. Effects of Temperature on Activity (A) and Stability (B) of Carboxypeptidase G 3 .
20.1
(A) Temperature-activity curve in 0.1 M phosphate buffer (pH 8.0) for 30 min of incubation. (B) Thermal stability curve. The enzyme was incubated at different temperatures in 0.1 M phosphate buffer (pH 8.0) for 30 min. The activities were assayed by method 1 as described in Materials and Methods.
14.4.
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Table II. Effects of Chemical Reagents and Inhibitors on Carboxypeptidase G 3 Activity
Lane
2
Fig. 2. SDS-Polyacrylamide Gel Electrophoresis of the Purified Carboxypeptidase G 3 . Electrophoresis of the purified enzyme and molecular markers was done on ExcelGel SDS, gradient 8-18. Lane I, M r standards: phosphorylase b (94,000); bovine serum albumin (67,000); ovalbumin (43,000); carbonic anhydrase (30,000); soybean trypsin inhibitor (20,100); a-lactalbumin (14,400). Lane 2, the purified enzyme.
....
100
A
B
\
~
....>. >
....u
/\
~
ell
50
~
>
.... ~
41
et::
5
6
7
8
None EGTA EGTA 0- Phenanthroline o-Phenanthroline N- Bromosuccinimide PCMB Iodoacetate PMSF DFP
5
9 10 11
Concentration (mM)
Residual activity (%)
0.1
100 29
1.0 0.1
35
3
1.0
8 6
1.0 1.0 1.0 1.0
81 94 100 94
1.0
The reaction mixture, 200 pI, consisting of enzyme (50 pI) and inhibitor at the given concentration in 50 mM phosphate buffer (pH 8) was incubated at 37°C for 20 min. The residual activity was assayed by method 1 as described in Materials and Methods. Table III.
/ 0
Chemical reagent
Effects of Metal Ions on Carboxypeptidase G 3 Activity Relative activity (%)
6
7
8
9 10 11
Metal ion (1 mM)
pH
Fig. 3. Effects of pH on Activity (A) and Stability (B) of Carboxypeptidase G 3 . (A) pH-Activity curve in the following buffers: 0, 0.1 M citrate-phosphate buffer (pH 5.0-6.0); .,0.1 M phosphate buffer (pH 6.0-8.0); 6,0.1 M Tris-HCI buffer (pH 8.0-9.0); ,A., 0.1 M carbonate buffer (pH 9.0-11.0). (B) pH-Stability curve. The enzyme was treated for 24 h at 4°C in the buffers (0.1 M) described in A. The activities were assayed by method 1 as described in Materials and Methods.
without these chelators. The inactivated enzyme was effectively activated by addition of Zn 2 +, slightly activated by Cu 2 +, Mn 2 +, and Co 2 +, and not affected by Ca 2 +, Fe 2 +, or Hg 2 + (Table III). Substrate specificity To investigate the specificity of the enzyme for the carboxyl-terminal amino acid, eleven kinds of N-octanoylamino acids were used as substrates. As shown in Table IV,
None CaCl 2 MnS0 4 FeS04 CoCl 2 Cu(CH 3 COOh ZnC1 2 HgC1 2
Native enzyme
EGTA treatment
100
2.4 4.9 29 5.2 13 54 94 0.7
66 19
6 14 4 157 0
The reaction mixture, 200 pI, consisting of enzyme (50 pI) and metal ion (1 mM) in 50 mM phosphate buffer (pH 8) was incubated at 37°C for 20 min. The activity was assayed by method 1 as described in Materials and Methods. The enzyme treated by metal ion chelating agent (20mM EGTA) was also assayed as described above.
N-octanoyl-DL-glutamic acid and -DL-aspartic acid were exclusively hydrolyzed, but the other N-octanoyl-amino acids were very slightly or not hydrolyzed. Unlike polymyxin
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Carboxypeptidase G 3 Table IV. Acids
Relative Activities of Carboxypeptidase G 3 for Acyl Amino 0.05
Substrate
Relative activity (%)
E c:
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C'l
Acetyl-(Cz)-oL-glutamic acid Butyroyl-(C4)-oL-glutamic acid Hexanoyl-(C 6)-oL-glutamic acid Octanoyl-(Cs)-oL-glutamic acid Decanoyl-(C1o)-oL-glutamic acid Dodecanoyl-(C1z)-OL-glutamic acid Tetradecaonyl-(C 14)-OL-glutamic acid Hexadecanoyl-(C 16 )-OL-glutamic acid Z-oL-glutamic acid Benzoyl-oL-glutamic acid Octanoyl-(Cs)-oL-aspartic acid Octanoyl-(Cs)-glycine Octanoyl-(Cs)-oL-alanine Octanoyl-(Cs)-oL-valine Octanoyl-(Cs)-oL-leucine Octanoyl-(Cs)-oL-serine Octanoyl-(Cs)-oL-threonine Octanoyl-(Cs)-oL-methionine Octanoyl-(Cs)-oL-phenylalanine Octanoyl-(C s)-6-aminohexanoic acid
38 42 81 100 III 50 39
The enzyme activity for octanoyl-(Cs)-oL-glutamic acid, corresponding to 33,umol/min/mg, was taken as 100%. Table V.
Substrate Specificities of Carboxypeptidase G 3 for Peptides
Substrate Octanoyl-Gly-Gly Octanoyl-Gly-Ala Octanoyl-Gly-Met Octanoyl-Gly-Leu Octanoyl-Ala-Gly Octanoyl-Gly-Gly-Gly-Gly Z-Gly-Gly Z-Gly-Phe Z-Ser-Ser-Tyr Z-Tyr-Gly-Gly Z-Asp(OBul)-Ala-Ala Z-Glu(OBul)-Ser-Thr-Leu Insulin B chain Poly-Glu (mean M r 5000) Poly-Asp (mean M r 5000) Z-Thr-Pro Z-Ser-Glu(OBul)-Lys(Boc)-Ser-Gln-Thr-Pro
iU
0.03
III
u
c:
(lJ
0,02
.0
0
