;. Biochem., 80, 39-43 (1976)
Further Studies on the Specificity of the Minor Ribonuclease
Masachika IRIE and Kazuko OHGI Department of Microbiology, Hoshi College of Pharmacy, Ebara, Shinagawa-ku, Tokyo 142 Received for publication, December 6, 1975
In order to investigate the base specificity of the minor RNase [EC 3.1. 4. 23] from Aspergillus saitoi, the kinetic constant of the enzyme was measured with 16 dinucleoside phosphates (XpY's) as substrates at pH 5.5 and 25°. The maximum rates of transesterification of GpY's were in the range of 10,000 to 2,800 and were markedly larger than those of other XpY's, including XpG's. The average Km values of UpY, CpY, ApY, and GpY increased in the order A, C, U, and G. This order coincides with that of the rates of release of 4 common nucleotides from RNA by RNase Ms (the rates decreased in the order 3'-GMP, 3'-AMP, 3'-CMP, and 3'-UMP), except for the case of GpY. Therefore the rates of release of nucleotides seem to be dependent on the affinity constant of the X base in XpY, except in the case of GpY. The high rate of release of guanylic acid from RNA was explained by the findings that higher rates of hydrolysis of GpY's compensate for their lower affinity to the enzyme. These results suggested that the base specificity was rather dependent upon the X nucleotide in XpY. The K\ values of various nucleotides and nucleosides towards RNase Ms were measured. These compounds inhibited the RNase competitively. Although the inhibitory effect depends on the bases, sugars and location of phosphate, when the location of phosphate on the sugar was the same, the Ki values of ribonucleotides decreased in the order U, G, C, and A and those of deoxyribonucleotides decreased in the order T, G, C, and A. The dependence of the inhibitory effect of ribonucleosides on the bases was similar to that of ribonucleotides, but that of deoxyribomicleosides was in the order dT, dA, dG, and dC.
It has been reported that two ribonucleases are produced by Aspergillus saitoi; they cleave the phosphodiester linkages between the nucleoside 3'-phosphate and 5'-hydroxyl group of the neighboring nucleoside of polynucleotides without strict base specificity (1, 2). The major component of the RNases from Aspergillus soitoi (RNase M) liberates 3'-AMP most Vol. 80, No. 1, 1976
39
rapidly from RNA among 4 possible 3'-nucleotides. The base specificity of this enzyme was studied by two means, cleavage of 16 dinucleoside phosphates (3) and inhibition of the enzyme with substrate analogs, nucleotides and nucleosides (4, 5). However, there is no information concerning the base specificity of the minor Aspergillus saitoi RNase (RNase Ms),
Downloaded from https://academic.oup.com/jb/article-abstract/80/1/39/770676 by university of winnipeg user on 18 January 2019
from Aspergillus saitoi
40
M. TRIE and K. OHGI
MATERIALS AND METHODS Enzyme—RNase Ms was obtained from a commercial digestive "Molsin" (Aspergillus saitoi) by the method of Ohgi and Irie (2). The enzyme preparation used was homogenous by disc electrophoresis on polyacrylamide gel. The enzyme concentration was determined from the absorbancy at 280 nm assuming that the absorbancy of a 0.1% solution was 1.7 (2). Substrate-Dinucleoside Phosphate (XpY)— Among 16 dinucleoside phosphates (XpY's, where X or Y represents one of the four main ribonucleoside residues of RNA, i.e., adenosine, guanosine, uridine, and cytidine) those having pyrimidine bases on the X side and GpC were synthesized by the method of Ukita et al. (6). GpG, GpA, ApC, ApG were purchased from Sigma (U.S.A.). ApA, ApU, GpU, 2', 3'-cyclic UMP and 2', 3'-cyclic CMP were obtained from Boehringer Manheim (Germany), and used without further purification. Nucleotides and Nucleosides—2'-AMP, 3'AMP, 2'-GMP, 3'-GMP, 3'- and 5'-deoxyribonucleotides, thymidine, and deoxycytidine were the products of Sigma Co. All 5'-ribonucleotides, 2',(3')-UMP, ribonucleosides, and 2'deoxyuridine were purchased from Kojin Co. Isomers of CMP, 2'-CMP, and 3'-CMP were separated from commercial 2', (3')-CMP by chromatography on a column of Dowex 1 (formate 1x8) according to the procedure of Cohn ( 7 ) . Measurement of Enzymatic Activity—(a) Dinucleoside phosphate as a substrate: The reaction mixture, consisting of 2.0 ml of 0.1 M
acetate buffer (pH 5.5) containing dinucleoside phosphate (38-95 //M) and 0.23-0.94 nmole of enzyme, was incubated at 25° and the reaction was followed by the increase in absorbance in the UV region in a Shimadzu UV spectrophotometer. The wavelength used to measure the enzymatic activity and the final absorbancy increments after complete hydrolysis of each XpY to Xp+Y were described in the previous paper (3). ( b) 2', 3'-Cyclic CMP as a substrate: The enzymatic activity of RNase Ms in the absence and presence of nucleotides and nucleosides was measured from the rate of formation of 3'-CMP from 2', 3'-cyclic CMP according to the methods of Richards (8). The reaction mixture (2 ml) consisted of 0.1 M acetate buffer (pH 5.5), 23-95 fiM 2', 3'-cyclic CMP, inhibitor and 0.94 nmole of enzyme. The reaction was followed at 286 nm with a Shimadzu UV 200 spectrophotometer. The concentrations of inhibitors used were 1—4 /iM for AMP and dAMP, 40-80 /iM for other nucleotides and 200 /JM for nucleosides. Determination of Kinetic Parameters—The Km and Fmn values were obtained from Lineweaver-Burk plots (9). The substrate and enzyme concentrations used were as described above. The reaction was carried out at pH 5.5 and 25°. The Ki values were also obtained by Lineweaver-Burk plots. RESULTS Cleavage of Dinucleoside Phosphates with RNase Ms—Since the hydrolysis of RNA with RNase is a rather complex reaction, it is difficult to analyze the exact significance of the kinetic parameters. Therefore, in order to investigate the base specificity of RNase Ms, the rates of cleavage of 16 dinucleoside phosphates were measured spectrophotometrically at 25° and pH 5.5. The optimum pH for the hydrolysis of RNA with RNase Ms was about 4.5 and that for the hydrolysis of cyclic CMP was 4.0. Most of the kinetic data for various RNases acting on XpY's have been measured in the pH range from 5.0 to 5.5. Therefore, though the rate of hydrolysis of 2',3'-cyclic CMP with RNase Ms at pH 5.5 / . Biochem.
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except that the RNase can liberate nucleotides from RNA in the order 3'-GMP, 3'-AMP, 3'CMP, and 3'-UMP. In the present paper, in order to study the precise base specificity of RNase Ms, the kinetic constants of the enzymatic cleavage of 16 dinucleoside phosphates by RNase Ms were measured. The inhibitory effects of various substrate analogs, nucleotides and nucleosides were also studied to obtain information on the contribution of the base, sugar and phosphate moieties of nucleotides to the affinity towards RNase Ms.
SPECIFICITY OF MINOR RIBONUCLEASE FROM Asp. saitoi TABLE I. Km and Vmar values of RNase Ms. The experimental conditions were as described in the text. Km (XlO^M)
ApA
0.23 0.37 0.49 0.31
ApG ApC ApU GpA GpG GpC GpU CpA CpG CpC CpU UpA UpG UpC UpU
2', 3'-Cyclic CMP 2',3'-Cyclic UMP
0.92 1.08 1.16 1.15
K m « (min-i) 8.4 7.8
19.8 10.3 3,660 10,200 9,200 2,840
0.29 0.35 0.71 0.27
10.0 15.2 14.2
0.36 0.24 0.45 0.66
24.0 19.6 12.4 22.6
1.6
14.0
4.3
5.7
4.4
was about 50% of that at pH 4.0 (optimal pH), it was decided to follow the transesterification reaction of XpY's at pH 5.5. The Michaelis constants and maximum velocities of RNase Ms towards 16 dinucleoside phosphates are listed in Table I. As can been seen from the table, most of the Michaelis constants were in the range of 20—70 pM, except for those of GpY's. In the cases of XpU and XpA, the Michaelis constants increased with change of X in the order C, A, U, and G, and A, C, U, and G, respectively. In the series of XpC and XpG, the Michaelis constants increased in the order U, A, C, and G, and U, C, A, and G, respectively. Thus, in every case, when X is the same, the Michaelis constants of XpY's are largest for the cases where Y is guanosine. In the cases where the nucleoside moiety on the 3' side of dinucleoside phosphate is uridine or adenosine, the Vm.x values of XpY decreased in the following order, G > U > C > A Vol. 80, No. 1, 1976
and G > U > A > C , respectively. When Y in XpY is cytidine or guanosine, Vm»i values decreased in the order G > A > C > U and G > U > C>A, respectively. In these cases, when X is guanosine, the Vmti values are approximately of the order of 104—105 min"1 and differ markedly from those of other dinucleoside phosphates where X is adenosine, uridine or cytidine. These results suggested that the base specificity was rather dependent upon the X bases in XpY. The average Km value of 4 dinucleoside phosphates having adenosine as the X moiety of XpY was smaller than those having the other three nucleosides as X. Average Km values of XpY's having cytidine, uridine, and guanosine as X followed that having adenosine in this order. These results indicate that the affinity of the base moiety towards RNase Ms decreased in the order A>C>U>G. Inhibition of RNase Ms with Various Nucleotides—The inhibitory effects of various ribonucleotides and deoxyribonucleotides towards RNase Ms were studied using 2', 3'-cyclic CMP as a substrate. All the nucleotides tested inhibited the enzymatic activity of RNase Ms competitively. The Ki values of the nucleotides are listed in Table II. The inhibitory effect depends upon the bases, sugars and the location of phosphate on the sugar. When the location of the phosphate moiety on the sugar was the same, the K\ values of ribonucleotides were in the order U > G > C > A and those of deoxyribonucleotides were in the order T > G > C > A . Thus, among nucleotides, the affinity of AMP towards RNase Ms was highest with both ribo- and deoxyribonucleotides, as well as with the XpY's described above. When the location of phosphate on the sugar was the same, the apparent affinity constants of ribonucleotides having a guanine base were between those having uracil (thymine for deoxyribonucleotides) and cytosine, the order being different from that observed with XpY's, where the Km value of GpY was the largest among ApY, CpY, and UpY. Inhibitory Effects of Various Nucleosides on RNase Ms—The inhibitory effects of various nucleosides on the enzymatic activity of RNase Ms were tested. The results are shown in
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Substrate
41
42
M. IRE and K. OHGI
TABLE II. K\ values of RNase Ms. The experimental conditions were a9 described in the text. DISCUSSION Inhibitor
Adenosine Guanosine Cytidine Uridine
Pi
0.27 0.68 1.7
20.0 10.0 6.5
Inhibitor
3'-dAMP 5'-dAMP 3'-dGMP 5'-dGMP
Ki (M X 10 5 )
0.24 3.2 3.1 4.8
3.0 1.8 6.5
31.0 45.0 4.5
15.0 6.1
37.0
3'-dCMP 5'-dCMP 3'-dTMP 5'-dTMP Deoxyadenosine Deoxyguanosine Deoxycytidine Deoxyuridine Thymidine
0.63 3.4 3.7 7.8
52.0 25.0 7.3
63.0 75.0
770.0
Table II. All the nucleosides tested inhibited the enzymatic activity of RNase Ms competitively. Although the Ki values of nucleosides having adenine as a base were larger than those of corresponding nucleotides having the same sugar moiety, in nucleosides having bases other than adenine, Ki values were not much different from those of nucleotides when the base was common. The K\ values of ribonucleosides were in the order A < C < G < U , and this order is the same as that of ribonucleotides described above. On the other hand, the affinity of deoxyribonucleosides decreased in the order dC, dG, dA, dU, and dT. The K\ value of dA was higher than those of dC and dG. The trend was quite different from those with ribonucleosides and nucleotides. It is not clear why the K\ value of dA is so much larger than that of dC or dG. However, when the base is the same, it seems reasonable to say that the Ki values of deoxyribonucleoside are larger than those of ribonucleosides.
The Vmax values of GpY's range between 3,000 to 10,000 and are markedly larger than those of the other 12 XpY's (about 10-20). However, in comparing the Km values of dinucleoside phosphates having the same base as Y, the Km values of GpY are larger than those of other XpY's, where X is C, U, or A. From these results, it appears that the specificity is determined mainly at the site where the X base in XpY is bound, and the site which binds the Y bases probably modifies the specificity a little. When RNA was hydrolyzed with RNase Ms, 3'-GMP was released first, followed by 3'AMP, 3'-CMP, and 3MJMP in that order. The preferential release of 3'-GMP could be explained by the higher transesterification rates of GpY, which more than compensate for the lower affinity of GpY's. The rates of release of other nucleotides (A, C, and U) cannot be explained simply by a comparison of the Km or Vmax values of ApY, GpY, and CpY as in the case of RNase M (3). The average Km values of UpY, CpY, ApY, and GpY are 43, 41, 37, and 107 fiM respectively, and they are thus in the order A < C < U < G . This order coincides with that of the rates of release of nucleotides from RNA by the action of RNase Ms, except for the case of GpY, which is cleaved too rapidly. Therefore, the rates of release of nucleotides seem to be dependent mainly on the affinity constant of the X base in XpY except when X is G. When X is G, a higher rate of hydrolysis compensates for the lower affinity, so GMP is released first. In contrast with the results in Table I, the binding constants of nucleotides and ribonucleosides change with change of the base in the order A > C > G > U . The Ki values can be considered theoretically to be equivalent to the affinity constant. Thus, the binding of nucleotides might be in the order of K\ values, i.e., the affinity of the GMP moiety for the enzyme lies between those of CMP and UMP. The difference between the order of the K\ values of nucleotides and that of the average Michaelis constants of GpY's, UpY's, CpY's, and ApY's /. Biochem.
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2'-AMP 3'-AMP 5'-AMP 2'-GMP 3'-GMP 5'-GMP 2'-CMP 3'-CMP 5'-CMP 2',(3')-UMP 5'-UMP
KI (M X 10B)
SPECIFICITY OF MINOR RIBONUCLEASE FROM Asp. saitoi
Vol. 80, No. 1, 1976
bility, a comparative study of the conformation of 3'-nucleotides and 3'-deoxyribonucleotides in solution may be necessary. The turnover numbers of RNase Ms for the 16 dinucleoside phosphates tested were of the order of 10 except in the cases of the 4 GpY's. These values are much smaller than those with RNase M, determined previously (3). In the latter case, turnover numbers were in the range of 2,000 to 12,000. These results might reflect the lower specific activity of RNase Ms as compared to RNase M (2). The authors thank Dr. Yoshida of Kikkoman Co. and Seishin Pharmaceutical Co. for the supply of "Molsin," the enzyme source. REFERENCES 1. Irie, M. (1967) / . Biochem. 62, 504 2. Ohgi, K. & Irie, M. (1975) / . Biochem. 77, 1085 3. Imazawa, M., Irie, M., & Ukita, T. (1968) / . Biochem. 64, 595 4. Irie, M. (1969) / . Biochem. 65, 133 5. Irie, M. (1969) / . Biochem. 65, 907 6. Ukita, T., Irie, M., Iraazawa, M., Furuichi, Y., Nishimura, H., & Sekiya, T. (1968) Seikagaku (in Japanese) 40, 363 7. Cohn, W.E. (1955) The Nucleic Acids (Davidson, J.N. & Chargaff, E.A., eds.) Vol. 1, pp. 230, Academic Press, New York 8. Richards, F.M. (1955) Compt. rend. trav. lab. Carlsberg ser. chim. 29, 315 9. Lineweaver, M. & Burk, D. (1934) / . Am. Chem. Soc. 56, 658 10. Ukita, T., Waku, K., Irie, M., & Hoshino, O. (1961) / . Biochem. 50, 405 11. Irie, M. (1964) / . Biochem. 56, 495
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might be explained if a higher turnover number of GpY increases the Km values somewhat; in this case, the estimated Km values for GpY's might be somewhat higher than the real affinity constants of the substrates. In the cases of RNase A [EC 3.1.4.22] (10), RNase T t [EC 3.1.4.8] ( / / ) , and RNase M [EC 3.1.4.23] {4), it was found that the inhibitory effects of the isomers of nucleotides are in the order 2'-nucleotides, 3'-nucleotides, and 5'-nucleotides. Therefore, the order seems to be general for RNases. However, in the case of RNase Ms, this tendency was found for AMP's and deoxyribonucleotides, but not for GMP and CMP. At present, it is not clear why this is so. Another peculiar phenomenon observed in this experiment was that the Ki values of ribonucleosides were lower than those of deoxyribonucleosides having the same base, but the K\ values of ribonucleotides were higher than those of deoxyribonucleotides having the same base. The former phenomenon was also observed with RNase M, and from the results, it was concluded that the hydroxyl groups of ribose are very important in the binding of inhibitor to the enzyme. However the observations described here seem to make such an argument ambiguous. On the other hand, it is possible that, in the case of nucleosides, the argument may be correct, while in 3'-deoxyribonucleotides, phosphate groups may be able to bind easily to the active site because there is no steric interaction between 2'-hydroxyl groups and phosphate, so the inhibitory effect of 3'-deoxyribonucleotides is more marked. To investigate this possi-
43
Further Studies on the Specificity of the Minor Ribonuclease
Masachika IRIE and Kazuko OHGI Department of Microbiology, Hoshi College of Pharmacy, Ebara, Shinagawa-ku, Tokyo 142 Received for publication, December 6, 1975
In order to investigate the base specificity of the minor RNase [EC 3.1. 4. 23] from Aspergillus saitoi, the kinetic constant of the enzyme was measured with 16 dinucleoside phosphates (XpY's) as substrates at pH 5.5 and 25°. The maximum rates of transesterification of GpY's were in the range of 10,000 to 2,800 and were markedly larger than those of other XpY's, including XpG's. The average Km values of UpY, CpY, ApY, and GpY increased in the order A, C, U, and G. This order coincides with that of the rates of release of 4 common nucleotides from RNA by RNase Ms (the rates decreased in the order 3'-GMP, 3'-AMP, 3'-CMP, and 3'-UMP), except for the case of GpY. Therefore the rates of release of nucleotides seem to be dependent on the affinity constant of the X base in XpY, except in the case of GpY. The high rate of release of guanylic acid from RNA was explained by the findings that higher rates of hydrolysis of GpY's compensate for their lower affinity to the enzyme. These results suggested that the base specificity was rather dependent upon the X nucleotide in XpY. The K\ values of various nucleotides and nucleosides towards RNase Ms were measured. These compounds inhibited the RNase competitively. Although the inhibitory effect depends on the bases, sugars and location of phosphate, when the location of phosphate on the sugar was the same, the Ki values of ribonucleotides decreased in the order U, G, C, and A and those of deoxyribonucleotides decreased in the order T, G, C, and A. The dependence of the inhibitory effect of ribonucleosides on the bases was similar to that of ribonucleotides, but that of deoxyribomicleosides was in the order dT, dA, dG, and dC.
It has been reported that two ribonucleases are produced by Aspergillus saitoi; they cleave the phosphodiester linkages between the nucleoside 3'-phosphate and 5'-hydroxyl group of the neighboring nucleoside of polynucleotides without strict base specificity (1, 2). The major component of the RNases from Aspergillus soitoi (RNase M) liberates 3'-AMP most Vol. 80, No. 1, 1976
39
rapidly from RNA among 4 possible 3'-nucleotides. The base specificity of this enzyme was studied by two means, cleavage of 16 dinucleoside phosphates (3) and inhibition of the enzyme with substrate analogs, nucleotides and nucleosides (4, 5). However, there is no information concerning the base specificity of the minor Aspergillus saitoi RNase (RNase Ms),
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from Aspergillus saitoi
40
M. TRIE and K. OHGI
MATERIALS AND METHODS Enzyme—RNase Ms was obtained from a commercial digestive "Molsin" (Aspergillus saitoi) by the method of Ohgi and Irie (2). The enzyme preparation used was homogenous by disc electrophoresis on polyacrylamide gel. The enzyme concentration was determined from the absorbancy at 280 nm assuming that the absorbancy of a 0.1% solution was 1.7 (2). Substrate-Dinucleoside Phosphate (XpY)— Among 16 dinucleoside phosphates (XpY's, where X or Y represents one of the four main ribonucleoside residues of RNA, i.e., adenosine, guanosine, uridine, and cytidine) those having pyrimidine bases on the X side and GpC were synthesized by the method of Ukita et al. (6). GpG, GpA, ApC, ApG were purchased from Sigma (U.S.A.). ApA, ApU, GpU, 2', 3'-cyclic UMP and 2', 3'-cyclic CMP were obtained from Boehringer Manheim (Germany), and used without further purification. Nucleotides and Nucleosides—2'-AMP, 3'AMP, 2'-GMP, 3'-GMP, 3'- and 5'-deoxyribonucleotides, thymidine, and deoxycytidine were the products of Sigma Co. All 5'-ribonucleotides, 2',(3')-UMP, ribonucleosides, and 2'deoxyuridine were purchased from Kojin Co. Isomers of CMP, 2'-CMP, and 3'-CMP were separated from commercial 2', (3')-CMP by chromatography on a column of Dowex 1 (formate 1x8) according to the procedure of Cohn ( 7 ) . Measurement of Enzymatic Activity—(a) Dinucleoside phosphate as a substrate: The reaction mixture, consisting of 2.0 ml of 0.1 M
acetate buffer (pH 5.5) containing dinucleoside phosphate (38-95 //M) and 0.23-0.94 nmole of enzyme, was incubated at 25° and the reaction was followed by the increase in absorbance in the UV region in a Shimadzu UV spectrophotometer. The wavelength used to measure the enzymatic activity and the final absorbancy increments after complete hydrolysis of each XpY to Xp+Y were described in the previous paper (3). ( b) 2', 3'-Cyclic CMP as a substrate: The enzymatic activity of RNase Ms in the absence and presence of nucleotides and nucleosides was measured from the rate of formation of 3'-CMP from 2', 3'-cyclic CMP according to the methods of Richards (8). The reaction mixture (2 ml) consisted of 0.1 M acetate buffer (pH 5.5), 23-95 fiM 2', 3'-cyclic CMP, inhibitor and 0.94 nmole of enzyme. The reaction was followed at 286 nm with a Shimadzu UV 200 spectrophotometer. The concentrations of inhibitors used were 1—4 /iM for AMP and dAMP, 40-80 /iM for other nucleotides and 200 /JM for nucleosides. Determination of Kinetic Parameters—The Km and Fmn values were obtained from Lineweaver-Burk plots (9). The substrate and enzyme concentrations used were as described above. The reaction was carried out at pH 5.5 and 25°. The Ki values were also obtained by Lineweaver-Burk plots. RESULTS Cleavage of Dinucleoside Phosphates with RNase Ms—Since the hydrolysis of RNA with RNase is a rather complex reaction, it is difficult to analyze the exact significance of the kinetic parameters. Therefore, in order to investigate the base specificity of RNase Ms, the rates of cleavage of 16 dinucleoside phosphates were measured spectrophotometrically at 25° and pH 5.5. The optimum pH for the hydrolysis of RNA with RNase Ms was about 4.5 and that for the hydrolysis of cyclic CMP was 4.0. Most of the kinetic data for various RNases acting on XpY's have been measured in the pH range from 5.0 to 5.5. Therefore, though the rate of hydrolysis of 2',3'-cyclic CMP with RNase Ms at pH 5.5 / . Biochem.
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except that the RNase can liberate nucleotides from RNA in the order 3'-GMP, 3'-AMP, 3'CMP, and 3'-UMP. In the present paper, in order to study the precise base specificity of RNase Ms, the kinetic constants of the enzymatic cleavage of 16 dinucleoside phosphates by RNase Ms were measured. The inhibitory effects of various substrate analogs, nucleotides and nucleosides were also studied to obtain information on the contribution of the base, sugar and phosphate moieties of nucleotides to the affinity towards RNase Ms.
SPECIFICITY OF MINOR RIBONUCLEASE FROM Asp. saitoi TABLE I. Km and Vmar values of RNase Ms. The experimental conditions were as described in the text. Km (XlO^M)
ApA
0.23 0.37 0.49 0.31
ApG ApC ApU GpA GpG GpC GpU CpA CpG CpC CpU UpA UpG UpC UpU
2', 3'-Cyclic CMP 2',3'-Cyclic UMP
0.92 1.08 1.16 1.15
K m « (min-i) 8.4 7.8
19.8 10.3 3,660 10,200 9,200 2,840
0.29 0.35 0.71 0.27
10.0 15.2 14.2
0.36 0.24 0.45 0.66
24.0 19.6 12.4 22.6
1.6
14.0
4.3
5.7
4.4
was about 50% of that at pH 4.0 (optimal pH), it was decided to follow the transesterification reaction of XpY's at pH 5.5. The Michaelis constants and maximum velocities of RNase Ms towards 16 dinucleoside phosphates are listed in Table I. As can been seen from the table, most of the Michaelis constants were in the range of 20—70 pM, except for those of GpY's. In the cases of XpU and XpA, the Michaelis constants increased with change of X in the order C, A, U, and G, and A, C, U, and G, respectively. In the series of XpC and XpG, the Michaelis constants increased in the order U, A, C, and G, and U, C, A, and G, respectively. Thus, in every case, when X is the same, the Michaelis constants of XpY's are largest for the cases where Y is guanosine. In the cases where the nucleoside moiety on the 3' side of dinucleoside phosphate is uridine or adenosine, the Vm.x values of XpY decreased in the following order, G > U > C > A Vol. 80, No. 1, 1976
and G > U > A > C , respectively. When Y in XpY is cytidine or guanosine, Vm»i values decreased in the order G > A > C > U and G > U > C>A, respectively. In these cases, when X is guanosine, the Vmti values are approximately of the order of 104—105 min"1 and differ markedly from those of other dinucleoside phosphates where X is adenosine, uridine or cytidine. These results suggested that the base specificity was rather dependent upon the X bases in XpY. The average Km value of 4 dinucleoside phosphates having adenosine as the X moiety of XpY was smaller than those having the other three nucleosides as X. Average Km values of XpY's having cytidine, uridine, and guanosine as X followed that having adenosine in this order. These results indicate that the affinity of the base moiety towards RNase Ms decreased in the order A>C>U>G. Inhibition of RNase Ms with Various Nucleotides—The inhibitory effects of various ribonucleotides and deoxyribonucleotides towards RNase Ms were studied using 2', 3'-cyclic CMP as a substrate. All the nucleotides tested inhibited the enzymatic activity of RNase Ms competitively. The Ki values of the nucleotides are listed in Table II. The inhibitory effect depends upon the bases, sugars and the location of phosphate on the sugar. When the location of the phosphate moiety on the sugar was the same, the K\ values of ribonucleotides were in the order U > G > C > A and those of deoxyribonucleotides were in the order T > G > C > A . Thus, among nucleotides, the affinity of AMP towards RNase Ms was highest with both ribo- and deoxyribonucleotides, as well as with the XpY's described above. When the location of phosphate on the sugar was the same, the apparent affinity constants of ribonucleotides having a guanine base were between those having uracil (thymine for deoxyribonucleotides) and cytosine, the order being different from that observed with XpY's, where the Km value of GpY was the largest among ApY, CpY, and UpY. Inhibitory Effects of Various Nucleosides on RNase Ms—The inhibitory effects of various nucleosides on the enzymatic activity of RNase Ms were tested. The results are shown in
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Substrate
41
42
M. IRE and K. OHGI
TABLE II. K\ values of RNase Ms. The experimental conditions were a9 described in the text. DISCUSSION Inhibitor
Adenosine Guanosine Cytidine Uridine
Pi
0.27 0.68 1.7
20.0 10.0 6.5
Inhibitor
3'-dAMP 5'-dAMP 3'-dGMP 5'-dGMP
Ki (M X 10 5 )
0.24 3.2 3.1 4.8
3.0 1.8 6.5
31.0 45.0 4.5
15.0 6.1
37.0
3'-dCMP 5'-dCMP 3'-dTMP 5'-dTMP Deoxyadenosine Deoxyguanosine Deoxycytidine Deoxyuridine Thymidine
0.63 3.4 3.7 7.8
52.0 25.0 7.3
63.0 75.0
770.0
Table II. All the nucleosides tested inhibited the enzymatic activity of RNase Ms competitively. Although the Ki values of nucleosides having adenine as a base were larger than those of corresponding nucleotides having the same sugar moiety, in nucleosides having bases other than adenine, Ki values were not much different from those of nucleotides when the base was common. The K\ values of ribonucleosides were in the order A < C < G < U , and this order is the same as that of ribonucleotides described above. On the other hand, the affinity of deoxyribonucleosides decreased in the order dC, dG, dA, dU, and dT. The K\ value of dA was higher than those of dC and dG. The trend was quite different from those with ribonucleosides and nucleotides. It is not clear why the K\ value of dA is so much larger than that of dC or dG. However, when the base is the same, it seems reasonable to say that the Ki values of deoxyribonucleoside are larger than those of ribonucleosides.
The Vmax values of GpY's range between 3,000 to 10,000 and are markedly larger than those of the other 12 XpY's (about 10-20). However, in comparing the Km values of dinucleoside phosphates having the same base as Y, the Km values of GpY are larger than those of other XpY's, where X is C, U, or A. From these results, it appears that the specificity is determined mainly at the site where the X base in XpY is bound, and the site which binds the Y bases probably modifies the specificity a little. When RNA was hydrolyzed with RNase Ms, 3'-GMP was released first, followed by 3'AMP, 3'-CMP, and 3MJMP in that order. The preferential release of 3'-GMP could be explained by the higher transesterification rates of GpY, which more than compensate for the lower affinity of GpY's. The rates of release of other nucleotides (A, C, and U) cannot be explained simply by a comparison of the Km or Vmax values of ApY, GpY, and CpY as in the case of RNase M (3). The average Km values of UpY, CpY, ApY, and GpY are 43, 41, 37, and 107 fiM respectively, and they are thus in the order A < C < U < G . This order coincides with that of the rates of release of nucleotides from RNA by the action of RNase Ms, except for the case of GpY, which is cleaved too rapidly. Therefore, the rates of release of nucleotides seem to be dependent mainly on the affinity constant of the X base in XpY except when X is G. When X is G, a higher rate of hydrolysis compensates for the lower affinity, so GMP is released first. In contrast with the results in Table I, the binding constants of nucleotides and ribonucleosides change with change of the base in the order A > C > G > U . The Ki values can be considered theoretically to be equivalent to the affinity constant. Thus, the binding of nucleotides might be in the order of K\ values, i.e., the affinity of the GMP moiety for the enzyme lies between those of CMP and UMP. The difference between the order of the K\ values of nucleotides and that of the average Michaelis constants of GpY's, UpY's, CpY's, and ApY's /. Biochem.
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2'-AMP 3'-AMP 5'-AMP 2'-GMP 3'-GMP 5'-GMP 2'-CMP 3'-CMP 5'-CMP 2',(3')-UMP 5'-UMP
KI (M X 10B)
SPECIFICITY OF MINOR RIBONUCLEASE FROM Asp. saitoi
Vol. 80, No. 1, 1976
bility, a comparative study of the conformation of 3'-nucleotides and 3'-deoxyribonucleotides in solution may be necessary. The turnover numbers of RNase Ms for the 16 dinucleoside phosphates tested were of the order of 10 except in the cases of the 4 GpY's. These values are much smaller than those with RNase M, determined previously (3). In the latter case, turnover numbers were in the range of 2,000 to 12,000. These results might reflect the lower specific activity of RNase Ms as compared to RNase M (2). The authors thank Dr. Yoshida of Kikkoman Co. and Seishin Pharmaceutical Co. for the supply of "Molsin," the enzyme source. REFERENCES 1. Irie, M. (1967) / . Biochem. 62, 504 2. Ohgi, K. & Irie, M. (1975) / . Biochem. 77, 1085 3. Imazawa, M., Irie, M., & Ukita, T. (1968) / . Biochem. 64, 595 4. Irie, M. (1969) / . Biochem. 65, 133 5. Irie, M. (1969) / . Biochem. 65, 907 6. Ukita, T., Irie, M., Iraazawa, M., Furuichi, Y., Nishimura, H., & Sekiya, T. (1968) Seikagaku (in Japanese) 40, 363 7. Cohn, W.E. (1955) The Nucleic Acids (Davidson, J.N. & Chargaff, E.A., eds.) Vol. 1, pp. 230, Academic Press, New York 8. Richards, F.M. (1955) Compt. rend. trav. lab. Carlsberg ser. chim. 29, 315 9. Lineweaver, M. & Burk, D. (1934) / . Am. Chem. Soc. 56, 658 10. Ukita, T., Waku, K., Irie, M., & Hoshino, O. (1961) / . Biochem. 50, 405 11. Irie, M. (1964) / . Biochem. 56, 495
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might be explained if a higher turnover number of GpY increases the Km values somewhat; in this case, the estimated Km values for GpY's might be somewhat higher than the real affinity constants of the substrates. In the cases of RNase A [EC 3.1.4.22] (10), RNase T t [EC 3.1.4.8] ( / / ) , and RNase M [EC 3.1.4.23] {4), it was found that the inhibitory effects of the isomers of nucleotides are in the order 2'-nucleotides, 3'-nucleotides, and 5'-nucleotides. Therefore, the order seems to be general for RNases. However, in the case of RNase Ms, this tendency was found for AMP's and deoxyribonucleotides, but not for GMP and CMP. At present, it is not clear why this is so. Another peculiar phenomenon observed in this experiment was that the Ki values of ribonucleosides were lower than those of deoxyribonucleosides having the same base, but the K\ values of ribonucleotides were higher than those of deoxyribonucleotides having the same base. The former phenomenon was also observed with RNase M, and from the results, it was concluded that the hydroxyl groups of ribose are very important in the binding of inhibitor to the enzyme. However the observations described here seem to make such an argument ambiguous. On the other hand, it is possible that, in the case of nucleosides, the argument may be correct, while in 3'-deoxyribonucleotides, phosphate groups may be able to bind easily to the active site because there is no steric interaction between 2'-hydroxyl groups and phosphate, so the inhibitory effect of 3'-deoxyribonucleotides is more marked. To investigate this possi-
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