/ . Biochem., 81, 1175-1180 (1977)
Junji HASHIMOTO 1 and Kenji TAKAHASHP Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, August 12, 1976
In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease Ui [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase Ut was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase Ti. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase Ui was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U t led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T x and Uj.
Ribonuclease Ux (RNase Uj) [ribonucleate 3'guanylo-oligonucleotidohydrolase, Ustilago sphaerogena, EC 3.1.4.8] is a guanine-specific ribonuclease, which was found in culture media of Ustilago sphaerogena (2). RNase \J1 is similar to RNase Tx in several respects, such as specificity, mode of action, molecular weight, amino acid composition (3-5) and immunological properties (6). 1
This study was supported in part by a grant (No. 868017) from the Ministry of Education, Science and Culture of Japan. A preliminary account of this study has appeared elsewhere (7). * Present address: Institute for Plant Virus Research, Aoba-cho, Chiba 280. • Present address: Department of Biochemistry, Primate Research Institute, Kyoto University, Inuyama, Aichi 484. To whom requests for reprints should be sent. Abbreviation: FDNB, l-fluorc-2,4-dinitrobenzene. Vol. 81, No. 4, 1977
1175
Amino acid sequence studies (7) have shown that the enzyme is homologous with RNase T t in primary structure and that it contains amino acid sequences corresponding to those in RNase T x involving His-40 and -92, Glu-58, and Arg-77, which have been implicated in the active site of the enzyme, although the sequence is different from that of RNase T x in about 60% of the total residues. Our previous results on the dye-sensitized photooxidation of RNase U! indicated the implication of one or two histidine residues in the active site of the enzyme (8), as in the case of RNase T t ( 9 11). In the present work, additional chemical modifications of RNase Ui were carried out in order to obtain further information on the nature of the amino acid residues in the active site of the enzyme. The present results indicate the presence, as in RNase Tu of a glutamic acid residue at the
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Chemical Modifications of Ribonuclease
J176
J. HASHIMOTO and K. TAKAHASHI
EXPERIMENTAL Materials—RNase L^ was prepared according to the procedure described in the previous paper (4). Performic acid-oxidized RNase U! was prepared by the procedure of Hirs (12). Pronase E was purchased from Kaken Chem. Co., Tokyo, and aminopeptidase M from Rohm and Haas Co., Philadelphia. Iodoacetic acid was obtained from Wako Pure Chem. Ind., Tokyo, and recrystallized from petroleum ether. [l-uC]-iodoacetic acid (13.9 mCi/mmol) was a product of New England Nuclear Co., Boston. Iodoacetamide was obtained from Wako Pure Chem. Ind., Tokyo, phenylglyoxal hydrate from K and K Laboratories, New York, and citraconic anhydride from Tokyo Kasei Kogyo Co., Tokyo. Determination of Enzymatic Activity—The enzymatic activity of RNase Uj was determined as described previously (13) by measuring the absorbance at 260 nm of the' acid-soluble digestion products obtained from yeast RNA, except that 0.2 M Tris-HCl buffer, pH 8.0, was used instead of 0.2 M Tris-HCl buffer, pH 7.5. Determination of Protein Concentration—Protein concentrations were determined from the absorbance at 280 nm, assuming ^2sonm = 15 for RNase U t . Amino Acid Analyses—Amino acids were determined with a Hitachi KLA-3B amino acid analyzer according to the procedure of Spackman et al. (14). Samples for analysis were prepared by hydrolysis in 1 ml of glass-distilled 6 N HO at 110°C for 24 h in evacuated, sealed tubes. Radioactivity Measurement—Radioactivity was measured with a Beckman LS-200B liquid scintillation counter in a vial containing 10-100 y\ of sample solution and 10 ml of a scintillation fluid (10 g of 2, 5-diphenyIoxazole, 0.25 g of bis [2'-(5'phenyloxazolyl)] benzene, and 100 g of recrystallized naphthalene in 1 liter of dioxane). Reaction of Iodoacetate—To prepare a reaction mixture, 0.3% RNase Ui in 0.2 M acetate buffer, fpH 5.5, was mixed with twice its volume of 3% J "C-labeled iodoacetate which had been dissolved
in water and brought to pH 5.5 with 1 N NaOH. The reaction was carried out in the dark at 37°C for 24 h. After the reaction, protein was freed from the reagents by passage of the reaction mixture through a column of Sephadex G-25 in the dark, eluting with 0.05 M acetic acid. Identification of the Amino Acid Residue Modified by Iodoacetate—After evaporation, 14Clabeled carboxymethylated RNase L^ (5 mg, 1.06 x 105cpm/^mol) was dissolved in 1.0 ml of 0.2 M Nethylmorpholine acetate buffer, pH 7.0, containing 0.5 mg of pronase. The mixture was kept at 37°C for 10 h and then brought to pH 1 with 6 N HC1. After keeping the mixture at 40°C for 15min, it was taken to dryness on a rotary evaporator and dissolved in 1.0 ml of 0.2 M N-ethylmorpholine acetate buffer, pH 7.0, containing 1.0 mg of aminopeptidase M. The mixture was kept at 37°C for 8 h. The digest was again evaporated to dryness, dissolved in 1.0 ml of 0.2 M citrate buffer, pH 2.2, and fractionated on a column (0.9 x 60 cm) of the amino acid analyzer. The effluent was diverted to a fraction collector before the ninhydnn reagent would normally enter the stream. Fractions of 2.0 ml were collected and 200- jA portions were withdrawn from each fraction for measurement of radioactivity. Radioactive fractions were pooled and subjected to acid hydrolysis and amino acid analysis. Reaction of Iodoacetamide—The reaction was performed at pH 8.0 in 0.1 M N-ethylmorpholine acetate buffer at 37°C in the dark at a protein concentration of 0.1 % and a reagent concentration of 1.5% for 24 h. After the reaction, the protein was freed from the reagents by passage through a Sephadex G-25 column in 0.01 M ammonium acetate. Reaction of Phenylglyoxal—To prepare a reaction mixture, a 0.1 % solution of RNase \Ji in 0 2 M N-ethylmorpholine acetate buffer, pH 8.0, was mixed with an equal volume of 3 % phenylglyoxal hydrate in the same buffer. The reaction mixture was kept at 25°C. After the reaction, protein was freed from the reagents by passage through a Sephadex G-25 column as described above. Reaction of Citraconic Anhydride—The reaction was performed at pH 8.0 in 0.2 M N-ethylmorpholine acetate buffer at 0°C at a protein concentration of 0.1% and a reagent concentration of 0.5 %. After the reaction, protein was freed from
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active site of RNase U! which is specifically modified by iodoacetate. The results also indicate that an arginine residue and a lysine residue may be located at or near the active site of the enzyme.
CHEMICAL MODIFICATIONS OF RNase U,
1177
RESULTS Stoichiometry of the Reaction of lodoacetate— When RNase Ux was treated with a large excess of iodoacetate at pH 5.5 and 37°C, enzymatic activity was lost at a rate comparable to that of the inactivation of RNase Tx (4). As can be seen in Table I, one carboxymethyl group per molecule of protein was shown to have been incorporated into the inactivated enzyme by the use of 14C-iodoacetate. Upon acid hydrolysis and amino acid analysis of the carboxymethylated enzyme, no loss of amino acid was observed. These results are identical with those obtained with RNase T^ indicating that an ester is formed at the carboxyl group of an aspartic or glutamic acid residue of RNase Ux. Identification of the Carboxymethylated Residue —In order to examine which amino acid in the enzyme, was carboxymethylated, the "C-labeled protein was digested successively with pronase and
aminopeptidase M, and the digest was analyzed by column chromatography. The digestion of native RNase Ux with these enzymes under the same conditions (see " EXPERIMENTAL") resulted in almost complete hydrolysis to free amino acids (more than 90% for each amino acid). The digest of the 14C-labeled protein (1.24xl0 4 cpm, 0.117 fimo\) was applied to a column (0.9x50 cm) on the amino acid analyzer. As can be seen in Fig. 1, elution with the first buffer for amino acid analysis gave a single radioactive peak (0.52x10* cpm) which emerged fairly well ahead of aspartic acid. Upon acid hydrolysis and amino acid analysis of the pooled radioactive fractions, only glutamic acid (0.03 pmol, overall yield 26%) could be detected as a ninhydrin-positive amino acid. The yield of glutamic acid was lower than expected from the radioactivity of the pooled fraction. This indicates that the radioactive fraction contained both 1*C-7"-carboxymethyl glutamic acid and 14Cglycolic acid liberated from it. The carboxy-
2000 -
1000 -
r TUBE NUMBER ( 2.0 ml / tube )
20
Fig. 1. Elution pattern of a pronase and aminopeptidase M digest of RNase Ux carboxymethylated with u C-labeled iodoacetate on a column (0.9 x 55 cm) of a Hitachi KLA-3B amino acid analyzer. Buffer: 0.2 N sodium citrate buffer, pH 3.25. Temperature: 57CC. Under these conditions, aspartic acid was eluted at about 50 ml effluent volume.
TABLE I. Number of carboxymethyl residues introduced into RNase U! by reaction with uC-labeled iodoacetate. The reaction was performed in 0.2 M acetate buffer, pH 5 5, at 37°C for 24 h at a protein concentration of 0.1% and a reagent concentration of 2%. Specific radioactivity Iodoacctic acid used Modified protein (cpm//imol) 1.26x10*
Vol. 81, No. 4, 1977
1.21x10*
Carboxymethyl residues introduced per molecule of protein
Remaining activity
0.96
0.1%
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the reagents by passage through a Sephadex G-25 column. The number of t-NH, groups of lysine residues which had reacted with citraconic anhydride was determined by amino acid analysis after dinitrophenylation (75). Measurement of Binding of3'-GMP to Modified RNase (//—The binding of 3'-GMP to modified RNase U1 was measured by the gel filtration method (16) as described previously (8). A column (0.9 x 20 cm) of Sephadex G-25 was equilibrated with 0.05 M sodium acetate buffer, pH 5.5, containing 0.066 mM 3'-GMP. About 0.6 mg of protein was dissolved in about 400 ptl of the same buffer containing 0.066 mM 3'-GMP. This solution was passed through the column at 25°C and 1.0-ml fractions were collected. The total protein eluted from the column was determined by amino acid analysis of an acid hydrolysate of a portion of the combined protein fraction.
1178
J. HASHIMOTO and K. TAKAHASHI
100
s 10 20 TIME OF REACTION ( h r )
Fig. 2. Rates of inactivation of RNase L1! by reaction with iodoacetamide, phenylglyoxal and citraconic anhydride. RNase Ut (0.1% solution) was treated with 1.5% iodoacetamide at pH 8.0 and 37°C ( • ) , with 1.5% phenylglyoxal hydrate at pH 8.0 and 25°C ( • ) or with 0.5% citraconic anhydride at 8 0 and 0°C (A).
Amino acid analysis of an acid hydrolysate of the inactivated protein showed the loss of about one residue each of histidine and lysine per molecule (Table II). Inactivation of RNase Ui by Phenylglyoxal— Phenylglyoxal has been developed as a reagent for the specific modification of arginine residues in proteins under mild conditions and has been successfully utilized for the modification of arginine residues in RNase A (18) and other proteins (1921). When RNase \JX was treated with a 1,100-fold molar excess of phenylglyoxal at pH 8.0 and 25°C, inactivation took place as shown in Fig. 2. Amino acid analysis of an acid hydrolysate of phenylglyoxal-treated RNase Ui (remaining activity, 2.2%) showed a loss of only about 1 residue of arginine (Table II). When RNase Ui was oxidized with performic acid prior to phenylglyoxal treatment, a slightly greater loss of arginine was observed (Table II). Phenylglyoxal-treated RNase Ui (remaining activity, 0.4%) was examined for binding ability toward 3'-GMP, a substrate analog,
TABLE II. Amino acid compositions of acid hydrolysates of some chemically modified derivatives of RNase U i . The values are given in terms of the molar ratios of amino acids assuming the number of lysine residues to be 3.0 for the phenylglyoxal-treated enzyme or the number of arginine residues to be 2.0 for the iodoacetamide-trcated enzyme and the citraconylated enzyme. The contents of other amino acids are not shown since there was no significant change in their contents, except for tyrosine in the citraconylated enzyme, which was lost completely upon dinitrophenylation and acid hydrolysis. The values in parentheses were obtained with performic acid-oxidized RNase U i . Amino acid
Theory for untreated enzyme (4)
Iodoacetamidctreated1
Phenylglyoxaltreated6
Citraconylated,0 then dinitrophenylated
2.0 0.9 2.0
3.0(3.0) 1.9(1.9) 0.7(0.6)
1.1(2.7) 0 (0 ) 2.0(2.0)
Lysine Histidine Arginine • Remaining activity, 10%. 3.7%. TABLE m .
b
Remaining activity, 2.2%.
» Remaining activity before dinitrophenylation,
Binding of 3'-GMP to native and chemically modified RNase Enzyme
RNase U, Citraconyl-RNase Uj Phenylglyoxal-treated RNase U x
Remaining activity (%) 100 2.6 0.4
Protein used (//mol) 0.052 0.059 0.061
3'-GMP bound to protein (fitno\) 0.032 0.039 0
0.036 0.036
J. Biochem.
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methylated residue of RNase L^ was thus concluded t o be glutamic acid, as in the case of RNase T x (17). Inactivation of RNase Ui by Iodoacetamide— Under the conditions employed, the enzyme was 90 % inactivated in 24 h, as can be seen from Fig. 2.
CHEMICAL MODIFICATIONS OF RNase U,
1179
Vol. 81, No. 4, 1977
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in the same way as RNase T! (17). The amino acid composition of an acid hydrolysate of the inactivated enzyme was indistinguishable from that of the native enzyme, indicating that the reaction took place at some specific residue to form an acidlabile derivative. Isolation and analysis of the 14Clabeled derivative indicated that the derivative is a 7-carboxymethyl ester of glutamic acid. The content of glutamic acid plus glutamine in RNase Ui is 6 residues per molecule, of which one is not 1 3 6 22 amidated, four are amidated, and one, in the NTIME OF REACTION ( hr ) terminal region, remains undetermined (7). AlFig. 3. Recovery of enzymatic activity of citraconylated though the location of the glutamic acid residue RNase U^ The modified enzyme (remaining activity, modified with iodoacetate in the amino acid se9.3%) was incubated at 25°C in ammonium formate quence of the enzyme has not been established, it buffer, pH 3.5. seems likely that this residue is the one in the sequence: -Glu-Tyr-Pro-Leu-Lys- (7). The seby the gel filtration method. The modified enzyme quence is homologous with that containing the had largely lost its binding ability, as shown in iodoacetate-reactive glutamic acid-58 in RNase Tj: Table HI. -Glu-Trp-Pro-Ile-Leu- (22). Thus, the inactivaInactivation of RNase Ui by Citraconic An- tion of RNase U is similar to that of RNase T in x x hydride—When the enzyme was treated with a 540- many respects, suggesting that the reactive glutamic fold molar excess of citraconic anhydride at pH 8.0 acid residue is a part of the active site of the enzyme, and 0°C, rapid inactivation took place, as shown as in RNase T . t in Fig. 2. Iodoacetamide inactivated the enzyme at pH The citraconylated RNase Ui (remaining 8.0 at a rate slightly higher than that observed with activity, 3.7%) was assayed for modified lysines. RNase T (23). The enzyme was inactivated with x The results of amino acid analysis of the derivatives simultaneous loss of about one residue each of after reaction with FDNB are shown in Table II. histidine and lysine per molecule. RNase Ux conCitraconylated lysine residues do not react with FDNB and are labile on acid hydrolysis. Hence, tains two histidine residues and both are thought the values of lysine residues given in Table II to be important for the activity (8). The results show the numbers of e-NH, groups modified. are thus consistent with this view. RNase Ux was also inactivated by reaction Table II shows that only 1 lysine residue was with phenylglyoxal, an arginine-specific reagent modified in citraconylated RNase Uj. When the enzyme was first oxidized with performic acid, all (18). The inactivation took place with concomithree lysine residues were modified (Table II). The tant modification of about one arginine residue out citraconylated RNase U t (remaining activity, 2.6%) of the two arginine residues per molecule. It retained almost the same binding ability toward 3'- seems quite likely that one arginine residue is crucial for the activity and is fairly specifically
Junji HASHIMOTO 1 and Kenji TAKAHASHP Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, August 12, 1976
In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease Ui [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase Ut was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase Ti. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase Ui was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U t led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T x and Uj.
Ribonuclease Ux (RNase Uj) [ribonucleate 3'guanylo-oligonucleotidohydrolase, Ustilago sphaerogena, EC 3.1.4.8] is a guanine-specific ribonuclease, which was found in culture media of Ustilago sphaerogena (2). RNase \J1 is similar to RNase Tx in several respects, such as specificity, mode of action, molecular weight, amino acid composition (3-5) and immunological properties (6). 1
This study was supported in part by a grant (No. 868017) from the Ministry of Education, Science and Culture of Japan. A preliminary account of this study has appeared elsewhere (7). * Present address: Institute for Plant Virus Research, Aoba-cho, Chiba 280. • Present address: Department of Biochemistry, Primate Research Institute, Kyoto University, Inuyama, Aichi 484. To whom requests for reprints should be sent. Abbreviation: FDNB, l-fluorc-2,4-dinitrobenzene. Vol. 81, No. 4, 1977
1175
Amino acid sequence studies (7) have shown that the enzyme is homologous with RNase T t in primary structure and that it contains amino acid sequences corresponding to those in RNase T x involving His-40 and -92, Glu-58, and Arg-77, which have been implicated in the active site of the enzyme, although the sequence is different from that of RNase T x in about 60% of the total residues. Our previous results on the dye-sensitized photooxidation of RNase U! indicated the implication of one or two histidine residues in the active site of the enzyme (8), as in the case of RNase T t ( 9 11). In the present work, additional chemical modifications of RNase Ui were carried out in order to obtain further information on the nature of the amino acid residues in the active site of the enzyme. The present results indicate the presence, as in RNase Tu of a glutamic acid residue at the
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Chemical Modifications of Ribonuclease
J176
J. HASHIMOTO and K. TAKAHASHI
EXPERIMENTAL Materials—RNase L^ was prepared according to the procedure described in the previous paper (4). Performic acid-oxidized RNase U! was prepared by the procedure of Hirs (12). Pronase E was purchased from Kaken Chem. Co., Tokyo, and aminopeptidase M from Rohm and Haas Co., Philadelphia. Iodoacetic acid was obtained from Wako Pure Chem. Ind., Tokyo, and recrystallized from petroleum ether. [l-uC]-iodoacetic acid (13.9 mCi/mmol) was a product of New England Nuclear Co., Boston. Iodoacetamide was obtained from Wako Pure Chem. Ind., Tokyo, phenylglyoxal hydrate from K and K Laboratories, New York, and citraconic anhydride from Tokyo Kasei Kogyo Co., Tokyo. Determination of Enzymatic Activity—The enzymatic activity of RNase Uj was determined as described previously (13) by measuring the absorbance at 260 nm of the' acid-soluble digestion products obtained from yeast RNA, except that 0.2 M Tris-HCl buffer, pH 8.0, was used instead of 0.2 M Tris-HCl buffer, pH 7.5. Determination of Protein Concentration—Protein concentrations were determined from the absorbance at 280 nm, assuming ^2sonm = 15 for RNase U t . Amino Acid Analyses—Amino acids were determined with a Hitachi KLA-3B amino acid analyzer according to the procedure of Spackman et al. (14). Samples for analysis were prepared by hydrolysis in 1 ml of glass-distilled 6 N HO at 110°C for 24 h in evacuated, sealed tubes. Radioactivity Measurement—Radioactivity was measured with a Beckman LS-200B liquid scintillation counter in a vial containing 10-100 y\ of sample solution and 10 ml of a scintillation fluid (10 g of 2, 5-diphenyIoxazole, 0.25 g of bis [2'-(5'phenyloxazolyl)] benzene, and 100 g of recrystallized naphthalene in 1 liter of dioxane). Reaction of Iodoacetate—To prepare a reaction mixture, 0.3% RNase Ui in 0.2 M acetate buffer, fpH 5.5, was mixed with twice its volume of 3% J "C-labeled iodoacetate which had been dissolved
in water and brought to pH 5.5 with 1 N NaOH. The reaction was carried out in the dark at 37°C for 24 h. After the reaction, protein was freed from the reagents by passage of the reaction mixture through a column of Sephadex G-25 in the dark, eluting with 0.05 M acetic acid. Identification of the Amino Acid Residue Modified by Iodoacetate—After evaporation, 14Clabeled carboxymethylated RNase L^ (5 mg, 1.06 x 105cpm/^mol) was dissolved in 1.0 ml of 0.2 M Nethylmorpholine acetate buffer, pH 7.0, containing 0.5 mg of pronase. The mixture was kept at 37°C for 10 h and then brought to pH 1 with 6 N HC1. After keeping the mixture at 40°C for 15min, it was taken to dryness on a rotary evaporator and dissolved in 1.0 ml of 0.2 M N-ethylmorpholine acetate buffer, pH 7.0, containing 1.0 mg of aminopeptidase M. The mixture was kept at 37°C for 8 h. The digest was again evaporated to dryness, dissolved in 1.0 ml of 0.2 M citrate buffer, pH 2.2, and fractionated on a column (0.9 x 60 cm) of the amino acid analyzer. The effluent was diverted to a fraction collector before the ninhydnn reagent would normally enter the stream. Fractions of 2.0 ml were collected and 200- jA portions were withdrawn from each fraction for measurement of radioactivity. Radioactive fractions were pooled and subjected to acid hydrolysis and amino acid analysis. Reaction of Iodoacetamide—The reaction was performed at pH 8.0 in 0.1 M N-ethylmorpholine acetate buffer at 37°C in the dark at a protein concentration of 0.1 % and a reagent concentration of 1.5% for 24 h. After the reaction, the protein was freed from the reagents by passage through a Sephadex G-25 column in 0.01 M ammonium acetate. Reaction of Phenylglyoxal—To prepare a reaction mixture, a 0.1 % solution of RNase \Ji in 0 2 M N-ethylmorpholine acetate buffer, pH 8.0, was mixed with an equal volume of 3 % phenylglyoxal hydrate in the same buffer. The reaction mixture was kept at 25°C. After the reaction, protein was freed from the reagents by passage through a Sephadex G-25 column as described above. Reaction of Citraconic Anhydride—The reaction was performed at pH 8.0 in 0.2 M N-ethylmorpholine acetate buffer at 0°C at a protein concentration of 0.1% and a reagent concentration of 0.5 %. After the reaction, protein was freed from
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active site of RNase U! which is specifically modified by iodoacetate. The results also indicate that an arginine residue and a lysine residue may be located at or near the active site of the enzyme.
CHEMICAL MODIFICATIONS OF RNase U,
1177
RESULTS Stoichiometry of the Reaction of lodoacetate— When RNase Ux was treated with a large excess of iodoacetate at pH 5.5 and 37°C, enzymatic activity was lost at a rate comparable to that of the inactivation of RNase Tx (4). As can be seen in Table I, one carboxymethyl group per molecule of protein was shown to have been incorporated into the inactivated enzyme by the use of 14C-iodoacetate. Upon acid hydrolysis and amino acid analysis of the carboxymethylated enzyme, no loss of amino acid was observed. These results are identical with those obtained with RNase T^ indicating that an ester is formed at the carboxyl group of an aspartic or glutamic acid residue of RNase Ux. Identification of the Carboxymethylated Residue —In order to examine which amino acid in the enzyme, was carboxymethylated, the "C-labeled protein was digested successively with pronase and
aminopeptidase M, and the digest was analyzed by column chromatography. The digestion of native RNase Ux with these enzymes under the same conditions (see " EXPERIMENTAL") resulted in almost complete hydrolysis to free amino acids (more than 90% for each amino acid). The digest of the 14C-labeled protein (1.24xl0 4 cpm, 0.117 fimo\) was applied to a column (0.9x50 cm) on the amino acid analyzer. As can be seen in Fig. 1, elution with the first buffer for amino acid analysis gave a single radioactive peak (0.52x10* cpm) which emerged fairly well ahead of aspartic acid. Upon acid hydrolysis and amino acid analysis of the pooled radioactive fractions, only glutamic acid (0.03 pmol, overall yield 26%) could be detected as a ninhydrin-positive amino acid. The yield of glutamic acid was lower than expected from the radioactivity of the pooled fraction. This indicates that the radioactive fraction contained both 1*C-7"-carboxymethyl glutamic acid and 14Cglycolic acid liberated from it. The carboxy-
2000 -
1000 -
r TUBE NUMBER ( 2.0 ml / tube )
20
Fig. 1. Elution pattern of a pronase and aminopeptidase M digest of RNase Ux carboxymethylated with u C-labeled iodoacetate on a column (0.9 x 55 cm) of a Hitachi KLA-3B amino acid analyzer. Buffer: 0.2 N sodium citrate buffer, pH 3.25. Temperature: 57CC. Under these conditions, aspartic acid was eluted at about 50 ml effluent volume.
TABLE I. Number of carboxymethyl residues introduced into RNase U! by reaction with uC-labeled iodoacetate. The reaction was performed in 0.2 M acetate buffer, pH 5 5, at 37°C for 24 h at a protein concentration of 0.1% and a reagent concentration of 2%. Specific radioactivity Iodoacctic acid used Modified protein (cpm//imol) 1.26x10*
Vol. 81, No. 4, 1977
1.21x10*
Carboxymethyl residues introduced per molecule of protein
Remaining activity
0.96
0.1%
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the reagents by passage through a Sephadex G-25 column. The number of t-NH, groups of lysine residues which had reacted with citraconic anhydride was determined by amino acid analysis after dinitrophenylation (75). Measurement of Binding of3'-GMP to Modified RNase (//—The binding of 3'-GMP to modified RNase U1 was measured by the gel filtration method (16) as described previously (8). A column (0.9 x 20 cm) of Sephadex G-25 was equilibrated with 0.05 M sodium acetate buffer, pH 5.5, containing 0.066 mM 3'-GMP. About 0.6 mg of protein was dissolved in about 400 ptl of the same buffer containing 0.066 mM 3'-GMP. This solution was passed through the column at 25°C and 1.0-ml fractions were collected. The total protein eluted from the column was determined by amino acid analysis of an acid hydrolysate of a portion of the combined protein fraction.
1178
J. HASHIMOTO and K. TAKAHASHI
100
s 10 20 TIME OF REACTION ( h r )
Fig. 2. Rates of inactivation of RNase L1! by reaction with iodoacetamide, phenylglyoxal and citraconic anhydride. RNase Ut (0.1% solution) was treated with 1.5% iodoacetamide at pH 8.0 and 37°C ( • ) , with 1.5% phenylglyoxal hydrate at pH 8.0 and 25°C ( • ) or with 0.5% citraconic anhydride at 8 0 and 0°C (A).
Amino acid analysis of an acid hydrolysate of the inactivated protein showed the loss of about one residue each of histidine and lysine per molecule (Table II). Inactivation of RNase Ui by Phenylglyoxal— Phenylglyoxal has been developed as a reagent for the specific modification of arginine residues in proteins under mild conditions and has been successfully utilized for the modification of arginine residues in RNase A (18) and other proteins (1921). When RNase \JX was treated with a 1,100-fold molar excess of phenylglyoxal at pH 8.0 and 25°C, inactivation took place as shown in Fig. 2. Amino acid analysis of an acid hydrolysate of phenylglyoxal-treated RNase Ui (remaining activity, 2.2%) showed a loss of only about 1 residue of arginine (Table II). When RNase Ui was oxidized with performic acid prior to phenylglyoxal treatment, a slightly greater loss of arginine was observed (Table II). Phenylglyoxal-treated RNase Ui (remaining activity, 0.4%) was examined for binding ability toward 3'-GMP, a substrate analog,
TABLE II. Amino acid compositions of acid hydrolysates of some chemically modified derivatives of RNase U i . The values are given in terms of the molar ratios of amino acids assuming the number of lysine residues to be 3.0 for the phenylglyoxal-treated enzyme or the number of arginine residues to be 2.0 for the iodoacetamide-trcated enzyme and the citraconylated enzyme. The contents of other amino acids are not shown since there was no significant change in their contents, except for tyrosine in the citraconylated enzyme, which was lost completely upon dinitrophenylation and acid hydrolysis. The values in parentheses were obtained with performic acid-oxidized RNase U i . Amino acid
Theory for untreated enzyme (4)
Iodoacetamidctreated1
Phenylglyoxaltreated6
Citraconylated,0 then dinitrophenylated
2.0 0.9 2.0
3.0(3.0) 1.9(1.9) 0.7(0.6)
1.1(2.7) 0 (0 ) 2.0(2.0)
Lysine Histidine Arginine • Remaining activity, 10%. 3.7%. TABLE m .
b
Remaining activity, 2.2%.
» Remaining activity before dinitrophenylation,
Binding of 3'-GMP to native and chemically modified RNase Enzyme
RNase U, Citraconyl-RNase Uj Phenylglyoxal-treated RNase U x
Remaining activity (%) 100 2.6 0.4
Protein used (//mol) 0.052 0.059 0.061
3'-GMP bound to protein (fitno\) 0.032 0.039 0
0.036 0.036
J. Biochem.
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methylated residue of RNase L^ was thus concluded t o be glutamic acid, as in the case of RNase T x (17). Inactivation of RNase Ui by Iodoacetamide— Under the conditions employed, the enzyme was 90 % inactivated in 24 h, as can be seen from Fig. 2.
CHEMICAL MODIFICATIONS OF RNase U,
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Vol. 81, No. 4, 1977
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in the same way as RNase T! (17). The amino acid composition of an acid hydrolysate of the inactivated enzyme was indistinguishable from that of the native enzyme, indicating that the reaction took place at some specific residue to form an acidlabile derivative. Isolation and analysis of the 14Clabeled derivative indicated that the derivative is a 7-carboxymethyl ester of glutamic acid. The content of glutamic acid plus glutamine in RNase Ui is 6 residues per molecule, of which one is not 1 3 6 22 amidated, four are amidated, and one, in the NTIME OF REACTION ( hr ) terminal region, remains undetermined (7). AlFig. 3. Recovery of enzymatic activity of citraconylated though the location of the glutamic acid residue RNase U^ The modified enzyme (remaining activity, modified with iodoacetate in the amino acid se9.3%) was incubated at 25°C in ammonium formate quence of the enzyme has not been established, it buffer, pH 3.5. seems likely that this residue is the one in the sequence: -Glu-Tyr-Pro-Leu-Lys- (7). The seby the gel filtration method. The modified enzyme quence is homologous with that containing the had largely lost its binding ability, as shown in iodoacetate-reactive glutamic acid-58 in RNase Tj: Table HI. -Glu-Trp-Pro-Ile-Leu- (22). Thus, the inactivaInactivation of RNase Ui by Citraconic An- tion of RNase U is similar to that of RNase T in x x hydride—When the enzyme was treated with a 540- many respects, suggesting that the reactive glutamic fold molar excess of citraconic anhydride at pH 8.0 acid residue is a part of the active site of the enzyme, and 0°C, rapid inactivation took place, as shown as in RNase T . t in Fig. 2. Iodoacetamide inactivated the enzyme at pH The citraconylated RNase Ui (remaining 8.0 at a rate slightly higher than that observed with activity, 3.7%) was assayed for modified lysines. RNase T (23). The enzyme was inactivated with x The results of amino acid analysis of the derivatives simultaneous loss of about one residue each of after reaction with FDNB are shown in Table II. histidine and lysine per molecule. RNase Ux conCitraconylated lysine residues do not react with FDNB and are labile on acid hydrolysis. Hence, tains two histidine residues and both are thought the values of lysine residues given in Table II to be important for the activity (8). The results show the numbers of e-NH, groups modified. are thus consistent with this view. RNase Ux was also inactivated by reaction Table II shows that only 1 lysine residue was with phenylglyoxal, an arginine-specific reagent modified in citraconylated RNase Uj. When the enzyme was first oxidized with performic acid, all (18). The inactivation took place with concomithree lysine residues were modified (Table II). The tant modification of about one arginine residue out citraconylated RNase U t (remaining activity, 2.6%) of the two arginine residues per molecule. It retained almost the same binding ability toward 3'- seems quite likely that one arginine residue is crucial for the activity and is fairly specifically
