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On the mechanism of action of Takadiastase ribonuclease T1
338
SHORT COMMUNICATIONS
1 T. Y. WANG, Arch. Biochera. Biophys., 97 (1962) 387 . a D. ELSON, Biochim. Biophys. Acta, 36 (1959) 372. a M. L. PETERMAN AND M. HAMILTON, in R. J. c. HARRIS, Protein Biosynthesis, A c a d e m i c Press~ L o n d o n a n d N e w York, 1961, p. 233. 4 M. TAKAI AND N. KONDO, BiocMm, B.iophys. Acta, 55 (1962)875. s U. Z. LITTAUER, in R. J. C. HARRIS, Protein Biosynthesis, A c a d e m i c Press, L o n d o n a n d N e w York, 1961, p. 143. 6 A. I. ARONSON AND B. J. McCARTHY, Biophys. J., i (1961) 215. G. K. HELMKAMP AND P. O. P. TS'O, Federation ProG., 19 (I96O) 316. 8 C. G. KURLAND, f . Mol. Biol., 2 (I96o) 83. * U. Z. LITTAUER AND H. EISENBERG, Biochim. Biophys. Aeta, 32 (I95
Received November I9th, 1962 Biochim. Biophys. Acta, 72 (1963) 335-338
sc 7o81
On the mechanism of action of Takadiastase ribonuclease Tz The cleavage of the 3',5'-diester bonds in ribonucleic acids by pancreatic ribonuclease (EC 2.7.7.16) and Takadiastase ribonuclease T 1 has been shown to follow the same reaction scheme 1, namely, a transesterification to a 2',3'-cyclic diester in the first step and a subsequent hydrolysis to 3'-monoesters in the second step. A strict specificity for different bases exists, however, for each enzyme. The kinetic results obtained by WITZEL AND BARNARD2 supported a mechanism proposed for pancreatic ribonuclease in which the pyrimidine base acts specifically on the catalysis and is not involved in the binding 3. The catalytic activity in the first step is enhanced considerably by the presence of the second nucleoside. Thus CpU is split 5 times faster, and CpA 500 times faster than cyclic cytidylic acid. If the same mechanism should hold for the T 1 enzyme, for which the function of the pyrimidine bases is replaced by that of the guanine base, then a similar enhancement of the reaction rates b y the second~ nucleoside would be expected. The observation 4 that hydrolysis of the cyclic diesters of oligonucleotides is faster than the hydrolysis of cyclic guanylic acid provides evidence for an influence of neighboring bases. We therefore sought to obtain some comparable data on the cleavage of various dinucleoside monophosphates by this enzyme. The compounds examined were 3'esters of guanylic, inosinic and xanthylic (I) acids and of a glyoxal derivative (II) of guanylic acid. It has been reported 5 that deaminated .RNA can be hydrolyzed by Biochim. Biophys. Acta, i1 (1963) 338-34 x
SHORT COMMUNICATIONS
339
ribonuClease T 1, the ester bonds of inosinic acid, derived from adenyric acid, being sprit quite readily, while those of xanthylic acid, derived from guanylic acid, are sprit much more slowly. The substrates GpCp, GpC, GpA, GpG, GpU and ApC were isolated from bismuth hydroxide hydrolysates of RNA e. XpC and IpC were derived from GpC and ApC by controlled deamination with nitrous acid. The glyoxal derivative of GpC was prepared by reaction of GpC with glyoxalv and isolated by paper chromatography in 70% isopropanol-water. Guanosine 2',3'-cyclic phosphate was prepared from guanyclic acid by the method of MICHELSON8 and purified by paper chromatography in 70% isopropanol-water with ammonia in the vapor phase 9. Ribonuclease Txwas very generously provided by Dr. G. W. RUSHIZKY,National Cancer Institute, National Institutes of Health, Bethesda, Md. (U.S.A.) The shift in the spectrum between guanosine 2',3'-cyclic phosphate and guanosine 3'-phosphate is not sufficient to provide a basis for a direct spectrophotometric assay of the enzyme, as used in the study of the pancreatic ribonuclease reaction s. Our results were obtained by the more cumbersome method of separating the products of the reaction by paper electrophoresis and then eluting and measuring the amount of each of the components in a spectrophotometer. Reactions were run in o.5-ml tubes and usually in groups of three: a no-enzyme control and two different enzyme concentrations, the latter being selected so that the amount of hydrolysis taking place in a 3o-min incubation at 37 ° would be in the range of lO-3O% of the substrate initially present. Electrophoresis on Whatman No. 3 MM paper was carried out at 18 V/cm in an apparatus similar to that described by MARKHAMAND SMITHs. With the exception of the separation of the nucleoside cyclic phosphates from the nucleoside 3'-phosphates which was done in o.05 M phosphate buffer (pH 7.4), all electrophoresis runs were carried out in o.o5 M formate buffer (pH 3.5). The amount of substrate hydrolyzed and the amount of one of the products formed were both measured and usually found to be in good agreement. The relative rates for the substrates tested are given in Table I. It can be seen TABLEI RELATIVE
RATES
OF SPLITTING OF DIFFERENT
Compound
SUBSTRATES
BY RIBONUCLEASE
T,
Relativerate*
GpCp
I I oo
GpC
800
GpA GpG GpU
55 ° 45° 250
IpC XpC
z5o io
Glyoxal-GpC
5
G-cyclle-P
2
" These values are the inverse of the amount o f e n z y m e (pg protein) required to hydrolyze o . o 2 / , m o l e of substrate in 30 rain a t 37 °. Reaction mixtures c o n t a i n e d , in a t o t a l v o l u m e of 3 °/~1, o.I 5 ?,mole of substrate, 0. 5/*mole Tris buffer ( p H 7.4) a n d 5/~1 of water or a p p r o p r i a t e l y diluted enzyme. After 3 ° rain a t 37 ° a 25-/,1 s a m p l e was subjected to electrophoresis and t h e amounts of the separated components determined.
Biochim. Biophys. Aaa, 72 (1963) 338-341
340
SHORT COMMUNICATIONS
that GpA is split at approximately twice the rate of GpU whereas the difference in the rate of splitting CpA and CpU by pancreatic ribonuclease is at least loo-fold (ref. 2). While ApC is completely resistant, the rate of splitting of IpC is 5 times lower and of XpC and the glyoxal-GpC is,80 and 16o.times lower than the rate for GpC. In the hydrolysis of !pC, glyoxal-GpC and XpC; the nucleoside cyclic phosphate, and not the 3'-phosphate, was always formed. These cyclic phosphate derivatives are very resistant to ribonuclease T 1 and, under conditions which resulted in 20% hydrolysis of guanosine cyclic phosphate, they were not hydrolyzed at all. The difference between cyclic guanylic acid and the slowest dinucleoside monophosphate, GpU, is more than a Ioo-fold, whereas the difference in the rates between cyclic cytidylic acid and CpU in the reaction of pancreatic ribonuclease is only 5-fold. Obviously the T1 enzyme shows a marked difference in its kinetic behavior compared with the pancreatic enzyme. In the first step there is only a small difference in the rates between the four dinucleoside phosphates. The enormous differences found in the reaction of pancreatic ribonuclease has been interpreted as a n-interaction between the two bases of the dinucleoside phosphates, thus increasing the nucleophilicity of th~ catalyzing pyrimidine base 2. Such an effect should be expected too, if the guanine base should play an analogous role in catalysis. Therefore, an identical type of catalysis cannot be assumed, in agreement with the previous statement" that a proton transfer from the f-hydroxyl group to the oxygen of the leaving alcohol cannot occur with the assistance of purine bases for steric reasons. The further supposition that the guanine base is specifically involved in the binding is supported by the observation that the reaction apparently requires the protonated form at N 1 (or perhaps at the oxygen in Position 6). Loss of the proton
OH
HOH0
0
rl ,,N
,,o..k,,,x,,,/ I
R
(I)
OH
1
> ,_,,,,,.k.,,,).,,,. H
I
I
R
(II)
I
R
(III)
R
(IV)
causes a marked decrease in the rates in the first step as well as in the second step. This agrees with the observation 1° that esters of 1-methyl guanylic acid (III) and the 2-dimethylamino compound (supposed in the Form IV where N 1 is blocked by the methyl group) are not split under conditions which cause a splitting of the guanylic acid esters. A final decision as to whether the guanine base is involved in the catalysis or in the binding will be possible only from a knowledge of the exact values for Km and the rate constants for the dinucleoside monophosphates and the cyclic phosphate. The authors are indebted to Mrs. E. FERCHE and Miss A. LEE for technical assistance. This investigation was supported in part by a U.S. Public Health Service training grant CRTY-5o28 from the National Cancer Institute, Public Health Service. Biochim. Biophys. Acta, 72 (I963) 338-341
341
SHORT COMMUNICATIONS
Virus Laboratory, University of California, Berkeley, Calif. (U.S.A.)
PAUL R. WHITFELD* H. WITZEL**
SATO AND F. EGAMI, J. Biochem., 44 (1957) 753. WITZEL AND E. A. BARNARD, Bioc~m. Biophy,. Res. Commun., 7 (1962) 289, 295. WITZEL, Ann. Chem., 635 (1960) I91. W . RUSHIZKY AND a . A. SOBER, J . Biol. Chem., 237 (1962) 834. 6 K . SATO-ASANO AND Y. FUJll, J. Biochem., 47 (196°) 6o8. ¢ K. DIMROTH AND H. WlTZEL, Ann. Chem., 62o (1959) lO9. "/ M. STAEHELIN,Biochim. Biophys. Acta, 31 (1959) 448. s A. M. MICHEL,SON, J. Chem. Sot., (1959) 3655 • 0 1:{. MARKHAM AND J. D. SMITH, Biochem. J., 52 (I952) 552. t0 K. S. McCuLLY AND G. L. CANTONI, Biochim. Biophys. Acta, 51 (1961) 19o. t K. t H. 8 H. 4 G.
Received January 2nd, 1963 * Fellow o f t h e Miller I n s t i t u t e for Basic R e s e a r c h in Science, o n leave o f a b s e n c e f r o m t h e Division o f P l a n t I n d u s t r y , C . S . I . R . 0 . , C a n b e r r a , A u s t r a l i a (present address). "" P r e s e n t a d d r e s s : C h e m i s c h e s I n s t i t u t d e r U n i v e r s i t ~ t M a r b u r g ( G e r m a n y ) .
Biochiin. Biophys. Aaa, 72 (1963) 338-341
SHORT COMMUNICATIONS
1 T. Y. WANG, Arch. Biochera. Biophys., 97 (1962) 387 . a D. ELSON, Biochim. Biophys. Acta, 36 (1959) 372. a M. L. PETERMAN AND M. HAMILTON, in R. J. c. HARRIS, Protein Biosynthesis, A c a d e m i c Press~ L o n d o n a n d N e w York, 1961, p. 233. 4 M. TAKAI AND N. KONDO, BiocMm, B.iophys. Acta, 55 (1962)875. s U. Z. LITTAUER, in R. J. C. HARRIS, Protein Biosynthesis, A c a d e m i c Press, L o n d o n a n d N e w York, 1961, p. 143. 6 A. I. ARONSON AND B. J. McCARTHY, Biophys. J., i (1961) 215. G. K. HELMKAMP AND P. O. P. TS'O, Federation ProG., 19 (I96O) 316. 8 C. G. KURLAND, f . Mol. Biol., 2 (I96o) 83. * U. Z. LITTAUER AND H. EISENBERG, Biochim. Biophys. Aeta, 32 (I95
Received November I9th, 1962 Biochim. Biophys. Acta, 72 (1963) 335-338
sc 7o81
On the mechanism of action of Takadiastase ribonuclease Tz The cleavage of the 3',5'-diester bonds in ribonucleic acids by pancreatic ribonuclease (EC 2.7.7.16) and Takadiastase ribonuclease T 1 has been shown to follow the same reaction scheme 1, namely, a transesterification to a 2',3'-cyclic diester in the first step and a subsequent hydrolysis to 3'-monoesters in the second step. A strict specificity for different bases exists, however, for each enzyme. The kinetic results obtained by WITZEL AND BARNARD2 supported a mechanism proposed for pancreatic ribonuclease in which the pyrimidine base acts specifically on the catalysis and is not involved in the binding 3. The catalytic activity in the first step is enhanced considerably by the presence of the second nucleoside. Thus CpU is split 5 times faster, and CpA 500 times faster than cyclic cytidylic acid. If the same mechanism should hold for the T 1 enzyme, for which the function of the pyrimidine bases is replaced by that of the guanine base, then a similar enhancement of the reaction rates b y the second~ nucleoside would be expected. The observation 4 that hydrolysis of the cyclic diesters of oligonucleotides is faster than the hydrolysis of cyclic guanylic acid provides evidence for an influence of neighboring bases. We therefore sought to obtain some comparable data on the cleavage of various dinucleoside monophosphates by this enzyme. The compounds examined were 3'esters of guanylic, inosinic and xanthylic (I) acids and of a glyoxal derivative (II) of guanylic acid. It has been reported 5 that deaminated .RNA can be hydrolyzed by Biochim. Biophys. Acta, i1 (1963) 338-34 x
SHORT COMMUNICATIONS
339
ribonuClease T 1, the ester bonds of inosinic acid, derived from adenyric acid, being sprit quite readily, while those of xanthylic acid, derived from guanylic acid, are sprit much more slowly. The substrates GpCp, GpC, GpA, GpG, GpU and ApC were isolated from bismuth hydroxide hydrolysates of RNA e. XpC and IpC were derived from GpC and ApC by controlled deamination with nitrous acid. The glyoxal derivative of GpC was prepared by reaction of GpC with glyoxalv and isolated by paper chromatography in 70% isopropanol-water. Guanosine 2',3'-cyclic phosphate was prepared from guanyclic acid by the method of MICHELSON8 and purified by paper chromatography in 70% isopropanol-water with ammonia in the vapor phase 9. Ribonuclease Txwas very generously provided by Dr. G. W. RUSHIZKY,National Cancer Institute, National Institutes of Health, Bethesda, Md. (U.S.A.) The shift in the spectrum between guanosine 2',3'-cyclic phosphate and guanosine 3'-phosphate is not sufficient to provide a basis for a direct spectrophotometric assay of the enzyme, as used in the study of the pancreatic ribonuclease reaction s. Our results were obtained by the more cumbersome method of separating the products of the reaction by paper electrophoresis and then eluting and measuring the amount of each of the components in a spectrophotometer. Reactions were run in o.5-ml tubes and usually in groups of three: a no-enzyme control and two different enzyme concentrations, the latter being selected so that the amount of hydrolysis taking place in a 3o-min incubation at 37 ° would be in the range of lO-3O% of the substrate initially present. Electrophoresis on Whatman No. 3 MM paper was carried out at 18 V/cm in an apparatus similar to that described by MARKHAMAND SMITHs. With the exception of the separation of the nucleoside cyclic phosphates from the nucleoside 3'-phosphates which was done in o.05 M phosphate buffer (pH 7.4), all electrophoresis runs were carried out in o.o5 M formate buffer (pH 3.5). The amount of substrate hydrolyzed and the amount of one of the products formed were both measured and usually found to be in good agreement. The relative rates for the substrates tested are given in Table I. It can be seen TABLEI RELATIVE
RATES
OF SPLITTING OF DIFFERENT
Compound
SUBSTRATES
BY RIBONUCLEASE
T,
Relativerate*
GpCp
I I oo
GpC
800
GpA GpG GpU
55 ° 45° 250
IpC XpC
z5o io
Glyoxal-GpC
5
G-cyclle-P
2
" These values are the inverse of the amount o f e n z y m e (pg protein) required to hydrolyze o . o 2 / , m o l e of substrate in 30 rain a t 37 °. Reaction mixtures c o n t a i n e d , in a t o t a l v o l u m e of 3 °/~1, o.I 5 ?,mole of substrate, 0. 5/*mole Tris buffer ( p H 7.4) a n d 5/~1 of water or a p p r o p r i a t e l y diluted enzyme. After 3 ° rain a t 37 ° a 25-/,1 s a m p l e was subjected to electrophoresis and t h e amounts of the separated components determined.
Biochim. Biophys. Aaa, 72 (1963) 338-341
340
SHORT COMMUNICATIONS
that GpA is split at approximately twice the rate of GpU whereas the difference in the rate of splitting CpA and CpU by pancreatic ribonuclease is at least loo-fold (ref. 2). While ApC is completely resistant, the rate of splitting of IpC is 5 times lower and of XpC and the glyoxal-GpC is,80 and 16o.times lower than the rate for GpC. In the hydrolysis of !pC, glyoxal-GpC and XpC; the nucleoside cyclic phosphate, and not the 3'-phosphate, was always formed. These cyclic phosphate derivatives are very resistant to ribonuclease T 1 and, under conditions which resulted in 20% hydrolysis of guanosine cyclic phosphate, they were not hydrolyzed at all. The difference between cyclic guanylic acid and the slowest dinucleoside monophosphate, GpU, is more than a Ioo-fold, whereas the difference in the rates between cyclic cytidylic acid and CpU in the reaction of pancreatic ribonuclease is only 5-fold. Obviously the T1 enzyme shows a marked difference in its kinetic behavior compared with the pancreatic enzyme. In the first step there is only a small difference in the rates between the four dinucleoside phosphates. The enormous differences found in the reaction of pancreatic ribonuclease has been interpreted as a n-interaction between the two bases of the dinucleoside phosphates, thus increasing the nucleophilicity of th~ catalyzing pyrimidine base 2. Such an effect should be expected too, if the guanine base should play an analogous role in catalysis. Therefore, an identical type of catalysis cannot be assumed, in agreement with the previous statement" that a proton transfer from the f-hydroxyl group to the oxygen of the leaving alcohol cannot occur with the assistance of purine bases for steric reasons. The further supposition that the guanine base is specifically involved in the binding is supported by the observation that the reaction apparently requires the protonated form at N 1 (or perhaps at the oxygen in Position 6). Loss of the proton
OH
HOH0
0
rl ,,N
,,o..k,,,x,,,/ I
R
(I)
OH
1
> ,_,,,,,.k.,,,).,,,. H
I
I
R
(II)
I
R
(III)
R
(IV)
causes a marked decrease in the rates in the first step as well as in the second step. This agrees with the observation 1° that esters of 1-methyl guanylic acid (III) and the 2-dimethylamino compound (supposed in the Form IV where N 1 is blocked by the methyl group) are not split under conditions which cause a splitting of the guanylic acid esters. A final decision as to whether the guanine base is involved in the catalysis or in the binding will be possible only from a knowledge of the exact values for Km and the rate constants for the dinucleoside monophosphates and the cyclic phosphate. The authors are indebted to Mrs. E. FERCHE and Miss A. LEE for technical assistance. This investigation was supported in part by a U.S. Public Health Service training grant CRTY-5o28 from the National Cancer Institute, Public Health Service. Biochim. Biophys. Acta, 72 (I963) 338-341
341
SHORT COMMUNICATIONS
Virus Laboratory, University of California, Berkeley, Calif. (U.S.A.)
PAUL R. WHITFELD* H. WITZEL**
SATO AND F. EGAMI, J. Biochem., 44 (1957) 753. WITZEL AND E. A. BARNARD, Bioc~m. Biophy,. Res. Commun., 7 (1962) 289, 295. WITZEL, Ann. Chem., 635 (1960) I91. W . RUSHIZKY AND a . A. SOBER, J . Biol. Chem., 237 (1962) 834. 6 K . SATO-ASANO AND Y. FUJll, J. Biochem., 47 (196°) 6o8. ¢ K. DIMROTH AND H. WlTZEL, Ann. Chem., 62o (1959) lO9. "/ M. STAEHELIN,Biochim. Biophys. Acta, 31 (1959) 448. s A. M. MICHEL,SON, J. Chem. Sot., (1959) 3655 • 0 1:{. MARKHAM AND J. D. SMITH, Biochem. J., 52 (I952) 552. t0 K. S. McCuLLY AND G. L. CANTONI, Biochim. Biophys. Acta, 51 (1961) 19o. t K. t H. 8 H. 4 G.
Received January 2nd, 1963 * Fellow o f t h e Miller I n s t i t u t e for Basic R e s e a r c h in Science, o n leave o f a b s e n c e f r o m t h e Division o f P l a n t I n d u s t r y , C . S . I . R . 0 . , C a n b e r r a , A u s t r a l i a (present address). "" P r e s e n t a d d r e s s : C h e m i s c h e s I n s t i t u t d e r U n i v e r s i t ~ t M a r b u r g ( G e r m a n y ) .
Biochiin. Biophys. Aaa, 72 (1963) 338-341