- Email: [email protected]
Peptidoasparaginase. An enzyme for deamidation of COOH-terminal peptide-bound asparagine
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSSCS
315-317
148,
(1972)
COMMUNICATIONS Peptidoasparaginase.
An Enzyme
for Deamidation
Recently L-asparaginase (L-Asparagine amidohydrolase, EC 3.5.1.1.) from microbial origin and guinea pig serum have been studied by a number of researchers for their antitumor activities (1, 2). The substrate specificity of previously rereported asparaginases is restricted to free asparagine and some compounds such as L-glutamine (1, 3). A purified L-asparaginase from guinea pig serum does not hydrolyze derivatives substituted at the a-amino group of asparagine, such as Nacetyl-L-asparagine, N-benzoyl-L-asparagine, and L-glutaminyl-L-asparagine (4). This indicates that the substitution of one hydrogen in the a-amino group of asparagine causes loss of activity. Halpern and Grossowicz showed that the asparaginase in crude extracts from Myco. phlei catalyzed the deamidation of glycyl-L-asparagine. But, this finding was due to contamination by a peptidase catalyzing the hydrolysis of the substrate into glycine and asparagine (5). During the course of studies on the purification of peptidoglutaminase from Bacillus circulans species, an activity deamidating the p-amide of CBZ-L-asparagine was detected (6). The fact that partially purified peptidoglutaminase-I and -11 did not catalyze the deamidation of CBZ-L-asparagine suggested the presence of another enzyme(s) which is specific for hydrolysis of the B-amide group of peptide-bound asparagine in the crude enzyme preparations. In t,his communication we describe the isolation and characteristics of a new asparaginase from B. circulans species and this enzyme has been named peptidoasparaginase because of the specificity for catalyzing the hydrolysis of the B-amide group of peptide-bound. asparagine. Reagents used were as follows: CBZ-I,-asparagine,’ CBZ-L-glutamine, and AOC-L-asparagine (Protein Research Foundation, Osaka, Japan); CBZ-nL-asparagine, CBZ-n-asparagine, BOC-L-asparagine, L-alanyl-L-asparagine, N-CBZ-L-alanyl-L-asparagine, and glycyl-L-glutamine (Cycle Chemical Corp.); glycyl-L-asparand glycyl-L-aspartic acid (Nutritional agine, Biochemical Corp.); AOC-L-glutaminyl+proline, 1 The following abbreviations are used: CBZ, N-tert-amyloxycarbenzyloxycarboxyl ; AOC, bonyl; BOC, IV-tert-butyloxycarbonyl. 315 Copyright
0
1972 by Academic
Press,
Inc.
of COOH-Terminal
Peptide-Bound
Asparagine
AOC-L-asparaginyl-L-proline, and L-alanyl-Lasparaginyl+phenylalanine (kindly supplied by Dr. S. Sakakibara of Protein Research Foundation, Osaka, Japan). A partial purification of the peptidoasparaginase was obtained by almost the same procedure as for peptidoglutaminase, with a slight modificaTABLE SUBSTRATE
SPECIFICITY
I
OF PEPTIDOASPARAGINASE’
Substrate
OD at 420 mp
Relative activity
CBZ-L-Asn CBZ-DL-Asn CBZ-n-Asn BOC-L-Asn AOC-L-Asn L-Ala-L-Asn Gly-L-Asn N-CBZ-L-Ala-L-Asn
0.556 0.474 0.080 0.329 0.533 0.499 0.527 0.410
1 0.852 0.143 0.589 0.959 0.897 0.948 0.737
L-Asn
0.323
0.581
AOC-L-Asn-L-Pro L-Ala-L-Asn-L-Phe
0.062 0
0.003 0
CBZ-L-Gln Gly-L-Gln AOC-L-Gln-L-Pro L-Gln
0.025 0.002 0.001 0.010
0.044 0.003 0.001 0.017
Q Incubation mixture: substrate, 10 pmoles; phosphate buffer (pH 7.6), 40 rmoles; enzyme 100 pg, all in 1.0 ml. Incubations carried out for 10 min at 30”. Under this experimental condition the reaction is linear for periods up to 20 min. Relative activity was calculated using CBZ-LAsn as a control. tion. All operations were done at O-6”. The reaction mixt.ure for peptidoasparaginase assay contained: 10 pmoles of CBZ-L-asparagine, 40 rmoles of phosphate buffer (pH 7.6) and suitably diluted enzyme solution, in a final volume of 2.0 ml. The reaction was for 10 min or 30 min at 30” and was stopped by the addition of %O vol of 5Ooj, trichloroacetic acid. The precipitated protein was removed
316
COMMUNICATIONS
by centrifugation, and the ammonia formed in the supernatant was determined by the direct addition of O-fold diluted Nessler’s reagent to 1.0 ml of the supernatant solution. Since CBZ-L-asparagine hydrolyzes in the very alkaline Nessler’s solution, the optical density at 420 rnM of the nessler color continues to increase. However, the rate of increase of optical density is essentially linear and slow enough (0.095 rmole/min/lO ml of 0.04 M CBZ-L-asparagine) that precisely timed blanks containing only CBZ-L-asparagine effectively cope with this problem. The ammonia content of the enzyme preparations was subtracted from these values. Cells of B. circulans sp. were suspended in 0.01 M phosphate buffer (pH 7.6) and homogenized with a Manton Gaulin homogenizer at an operating pressure of 7000 psi. The homogenized material was centrifuged for 90 min at 50,OOOg.The resulting supernatant fluid (crude extract) was collected and streptomycin sulfate was added until precipitation was complete. The supernatant fluid was fractionated with solid ammonium sulfate and the precipitate obtained between 35y0 and 50% saturation was dissolved in a minimum amount of 0.01 M phosphate buffer (pH 7.6). The sample was applied to a column 2 X 159 cm
- 0.8 xfI 0.6 fc 6
of Sephadex G-200 previously equilibrated with 0.01 M phosphate buffer (pH 7.6) containing 0.1 M KCl, and the column was developed with the same buffer. Each 4.8~ml fraction was collected. The activity of the enzyme which catalyzes the hydrolysis of the B-amide of CBZ-L-asparagine was detected in fraction No. 44 and that of peptidoglutaminases at No. 51 and 54 (Fig. 1). By this gel filtration, peptidoasparaginase was well separated from peptidoglutaminase-I and -11. Fractions 4046 were pooled and concentrated to x0 vol in a collodion bag, and the experiments in this paper were conducted on this fraction. Table I shows the substrate specificity of peptidoasparaginase. This enzyme hydrolyzed the pamide of free asparagine. When the or-amino group was involved in a peptide bond, hydrolysis of the P-amide occurred at about twice the rate, except with CBZ-n-asparagine and BOC-L-asparagine. A derivative substituted at both the carboxyl group and the a-amino group, such as AOC-Lasparaginyl-L-proline was not attacked. Though CBZ-L-glutamine is slightly hydrolyzed, this might be caused by contamination with small amounts of peptidoglutaminases in this preparation.
CBZ-AsN
0.4
d
0.2
20
40
60
80
FIG. 1. Gel filtration of Peptidoasparaginase on Sephadex G-200. Approximately 50 mg protein of the 35-w0 ammonium sulfate fraction was applied to a Sephadex G-200 column (2 X 150 cm) and eluted with the phosphate buffer containing 0.1 M KC1 at a rate of 16 ml/hr. Fractions of 4.8 ml were collected and 1.0 ml from each fraction was assayed for asparaginase activity. Incubation was 10 min at 30’. Incubation mixtures of peptidoglutaminase contained: 10 pmoles of CBZ-L-glutamine (for peptidoglutaminase-I) or AOC-L-Gln-L-Pro (for peptidoglutaminase-II), 40 amoles of phosphate buffer @H 7.5), CBZ-L-&n; --@[email protected] ml of each fraction. Incubation was 10 min at 30”. --@--oAOC-L-Gln-L-Pro. CBZ-x.-Gln; -X--X-
317
COMMUNICATIONS These results indicate that this peptidoasparaginase is specific for the hydrolysis of both the 8amide of asparagine residues which are located at the carboxyl-terminal end of a peptide and that of free asparagine. Moreover, the absence of any contaminating peptidase activity in these specificity experiments was ensured by thin-layer chromatography and paper electrophoresis. When glycyl-Lasparagine was incubated with peptidoasparaginase, glycyl-L-aspartic acid was produced, but no free glycine, aspartic acid, or asparagine was detected. It is clear, therefore, that the enzyme attacks the intact peptide. The results presented here show that there exist a novel enzyme catalyzing the deamidation of COOH-terminal peptide-bound asparagine in the microbia.1 extracts. ACKNOWLEDGMENTS
REFERENCES 1. WRISTON, J. C., JR., Methods Enzymol.
17, 732
(1970). 2. CROWTHER, D., Nature London 229, 168 (1971). 3. RAMADAN, M. E. A., EL ASMAR, E., AND GRIENBERQ, D. M., Arch. Biochem. Biophys. 108, 143 (1964). 4. TOWER, D. B., PETERS, E. L., AND CURTIS, W. C., J. Biol. Chem. 288,983 (1963). 5. HALPERN, Y. S., AND GROSSOWICZ, N., Biochem. J. 66, 716 (1957). 6. KIKUCHI, M., HAYASHIDA, H., NAK.~NO, E., AND SAKAGUCHI, K., Biochemistry 10, 1222 (1971). MAMORU KIKUCHI KENJI SAKAC~UCHI~ Research
Noda Institute for Scientific Noda-Shi, Chiba-Ken, Japan Received August 23, 1071; accepted September 89,
1971 We are indebted to Professor S. Sakakibara for advice and for synthesizing peptides. We are grateful to Professors Y. Ikeda and K. Arima for valuable advice.
* Present address: Mitsubishi-Kasei Institute of Life Science, y. Mitsubishi Chemical Industry, Limited, Marunouchi, Chiyodaku, Tokyo, Japan.
OF
BIOCHEMISTRY
AND
BIOPHYSSCS
315-317
148,
(1972)
COMMUNICATIONS Peptidoasparaginase.
An Enzyme
for Deamidation
Recently L-asparaginase (L-Asparagine amidohydrolase, EC 3.5.1.1.) from microbial origin and guinea pig serum have been studied by a number of researchers for their antitumor activities (1, 2). The substrate specificity of previously rereported asparaginases is restricted to free asparagine and some compounds such as L-glutamine (1, 3). A purified L-asparaginase from guinea pig serum does not hydrolyze derivatives substituted at the a-amino group of asparagine, such as Nacetyl-L-asparagine, N-benzoyl-L-asparagine, and L-glutaminyl-L-asparagine (4). This indicates that the substitution of one hydrogen in the a-amino group of asparagine causes loss of activity. Halpern and Grossowicz showed that the asparaginase in crude extracts from Myco. phlei catalyzed the deamidation of glycyl-L-asparagine. But, this finding was due to contamination by a peptidase catalyzing the hydrolysis of the substrate into glycine and asparagine (5). During the course of studies on the purification of peptidoglutaminase from Bacillus circulans species, an activity deamidating the p-amide of CBZ-L-asparagine was detected (6). The fact that partially purified peptidoglutaminase-I and -11 did not catalyze the deamidation of CBZ-L-asparagine suggested the presence of another enzyme(s) which is specific for hydrolysis of the B-amide group of peptide-bound asparagine in the crude enzyme preparations. In t,his communication we describe the isolation and characteristics of a new asparaginase from B. circulans species and this enzyme has been named peptidoasparaginase because of the specificity for catalyzing the hydrolysis of the B-amide group of peptide-bound. asparagine. Reagents used were as follows: CBZ-I,-asparagine,’ CBZ-L-glutamine, and AOC-L-asparagine (Protein Research Foundation, Osaka, Japan); CBZ-nL-asparagine, CBZ-n-asparagine, BOC-L-asparagine, L-alanyl-L-asparagine, N-CBZ-L-alanyl-L-asparagine, and glycyl-L-glutamine (Cycle Chemical Corp.); glycyl-L-asparand glycyl-L-aspartic acid (Nutritional agine, Biochemical Corp.); AOC-L-glutaminyl+proline, 1 The following abbreviations are used: CBZ, N-tert-amyloxycarbenzyloxycarboxyl ; AOC, bonyl; BOC, IV-tert-butyloxycarbonyl. 315 Copyright
0
1972 by Academic
Press,
Inc.
of COOH-Terminal
Peptide-Bound
Asparagine
AOC-L-asparaginyl-L-proline, and L-alanyl-Lasparaginyl+phenylalanine (kindly supplied by Dr. S. Sakakibara of Protein Research Foundation, Osaka, Japan). A partial purification of the peptidoasparaginase was obtained by almost the same procedure as for peptidoglutaminase, with a slight modificaTABLE SUBSTRATE
SPECIFICITY
I
OF PEPTIDOASPARAGINASE’
Substrate
OD at 420 mp
Relative activity
CBZ-L-Asn CBZ-DL-Asn CBZ-n-Asn BOC-L-Asn AOC-L-Asn L-Ala-L-Asn Gly-L-Asn N-CBZ-L-Ala-L-Asn
0.556 0.474 0.080 0.329 0.533 0.499 0.527 0.410
1 0.852 0.143 0.589 0.959 0.897 0.948 0.737
L-Asn
0.323
0.581
AOC-L-Asn-L-Pro L-Ala-L-Asn-L-Phe
0.062 0
0.003 0
CBZ-L-Gln Gly-L-Gln AOC-L-Gln-L-Pro L-Gln
0.025 0.002 0.001 0.010
0.044 0.003 0.001 0.017
Q Incubation mixture: substrate, 10 pmoles; phosphate buffer (pH 7.6), 40 rmoles; enzyme 100 pg, all in 1.0 ml. Incubations carried out for 10 min at 30”. Under this experimental condition the reaction is linear for periods up to 20 min. Relative activity was calculated using CBZ-LAsn as a control. tion. All operations were done at O-6”. The reaction mixt.ure for peptidoasparaginase assay contained: 10 pmoles of CBZ-L-asparagine, 40 rmoles of phosphate buffer (pH 7.6) and suitably diluted enzyme solution, in a final volume of 2.0 ml. The reaction was for 10 min or 30 min at 30” and was stopped by the addition of %O vol of 5Ooj, trichloroacetic acid. The precipitated protein was removed
316
COMMUNICATIONS
by centrifugation, and the ammonia formed in the supernatant was determined by the direct addition of O-fold diluted Nessler’s reagent to 1.0 ml of the supernatant solution. Since CBZ-L-asparagine hydrolyzes in the very alkaline Nessler’s solution, the optical density at 420 rnM of the nessler color continues to increase. However, the rate of increase of optical density is essentially linear and slow enough (0.095 rmole/min/lO ml of 0.04 M CBZ-L-asparagine) that precisely timed blanks containing only CBZ-L-asparagine effectively cope with this problem. The ammonia content of the enzyme preparations was subtracted from these values. Cells of B. circulans sp. were suspended in 0.01 M phosphate buffer (pH 7.6) and homogenized with a Manton Gaulin homogenizer at an operating pressure of 7000 psi. The homogenized material was centrifuged for 90 min at 50,OOOg.The resulting supernatant fluid (crude extract) was collected and streptomycin sulfate was added until precipitation was complete. The supernatant fluid was fractionated with solid ammonium sulfate and the precipitate obtained between 35y0 and 50% saturation was dissolved in a minimum amount of 0.01 M phosphate buffer (pH 7.6). The sample was applied to a column 2 X 159 cm
- 0.8 xfI 0.6 fc 6
of Sephadex G-200 previously equilibrated with 0.01 M phosphate buffer (pH 7.6) containing 0.1 M KCl, and the column was developed with the same buffer. Each 4.8~ml fraction was collected. The activity of the enzyme which catalyzes the hydrolysis of the B-amide of CBZ-L-asparagine was detected in fraction No. 44 and that of peptidoglutaminases at No. 51 and 54 (Fig. 1). By this gel filtration, peptidoasparaginase was well separated from peptidoglutaminase-I and -11. Fractions 4046 were pooled and concentrated to x0 vol in a collodion bag, and the experiments in this paper were conducted on this fraction. Table I shows the substrate specificity of peptidoasparaginase. This enzyme hydrolyzed the pamide of free asparagine. When the or-amino group was involved in a peptide bond, hydrolysis of the P-amide occurred at about twice the rate, except with CBZ-n-asparagine and BOC-L-asparagine. A derivative substituted at both the carboxyl group and the a-amino group, such as AOC-Lasparaginyl-L-proline was not attacked. Though CBZ-L-glutamine is slightly hydrolyzed, this might be caused by contamination with small amounts of peptidoglutaminases in this preparation.
CBZ-AsN
0.4
d
0.2
20
40
60
80
FIG. 1. Gel filtration of Peptidoasparaginase on Sephadex G-200. Approximately 50 mg protein of the 35-w0 ammonium sulfate fraction was applied to a Sephadex G-200 column (2 X 150 cm) and eluted with the phosphate buffer containing 0.1 M KC1 at a rate of 16 ml/hr. Fractions of 4.8 ml were collected and 1.0 ml from each fraction was assayed for asparaginase activity. Incubation was 10 min at 30’. Incubation mixtures of peptidoglutaminase contained: 10 pmoles of CBZ-L-glutamine (for peptidoglutaminase-I) or AOC-L-Gln-L-Pro (for peptidoglutaminase-II), 40 amoles of phosphate buffer @H 7.5), CBZ-L-&n; --@[email protected] ml of each fraction. Incubation was 10 min at 30”. --@--oAOC-L-Gln-L-Pro. CBZ-x.-Gln; -X--X-
317
COMMUNICATIONS These results indicate that this peptidoasparaginase is specific for the hydrolysis of both the 8amide of asparagine residues which are located at the carboxyl-terminal end of a peptide and that of free asparagine. Moreover, the absence of any contaminating peptidase activity in these specificity experiments was ensured by thin-layer chromatography and paper electrophoresis. When glycyl-Lasparagine was incubated with peptidoasparaginase, glycyl-L-aspartic acid was produced, but no free glycine, aspartic acid, or asparagine was detected. It is clear, therefore, that the enzyme attacks the intact peptide. The results presented here show that there exist a novel enzyme catalyzing the deamidation of COOH-terminal peptide-bound asparagine in the microbia.1 extracts. ACKNOWLEDGMENTS
REFERENCES 1. WRISTON, J. C., JR., Methods Enzymol.
17, 732
(1970). 2. CROWTHER, D., Nature London 229, 168 (1971). 3. RAMADAN, M. E. A., EL ASMAR, E., AND GRIENBERQ, D. M., Arch. Biochem. Biophys. 108, 143 (1964). 4. TOWER, D. B., PETERS, E. L., AND CURTIS, W. C., J. Biol. Chem. 288,983 (1963). 5. HALPERN, Y. S., AND GROSSOWICZ, N., Biochem. J. 66, 716 (1957). 6. KIKUCHI, M., HAYASHIDA, H., NAK.~NO, E., AND SAKAGUCHI, K., Biochemistry 10, 1222 (1971). MAMORU KIKUCHI KENJI SAKAC~UCHI~ Research
Noda Institute for Scientific Noda-Shi, Chiba-Ken, Japan Received August 23, 1071; accepted September 89,
1971 We are indebted to Professor S. Sakakibara for advice and for synthesizing peptides. We are grateful to Professors Y. Ikeda and K. Arima for valuable advice.
* Present address: Mitsubishi-Kasei Institute of Life Science, y. Mitsubishi Chemical Industry, Limited, Marunouchi, Chiyodaku, Tokyo, Japan.