[32]
R I B O N U C L E O S I DDIPHOSPHATE E REDUCTASE
321
sary when long (12 hr) incubations are employed. For example, at pH 7 and 0 °, the half-life of Co-(phen)-ATP was found to be 20 hr. The stoichiometry obtained both in the case of myosin and its subfragments 3,* and in the case of coupling factor 14,7 indicates that the labeling occurs only at specific sites on the enzymes. Moreover, the fact that it is possible to separate the labeled enzymes from excess reagent is by itself a proof that the association with the complex is irreversible. Co-(phen)-ATP was also shown to behave as a competitive inhibitor of myosin a and of coupling factor 1 when added directly into the assay medium. 8 Moreover, in the case of heavy meromyosin, both Mg ATP and Mg ADP could protect against labeling by Co-(phen)-ATP. * Experiments with thiol reagents (p-hydroxymercuribenzoate and N-ethylmaleimide) seem to indicate that Co-(phen)-ATP either binds to or protects some essential SH groups in the active site of myosin. The structure of Co-(phen)-ATP has been investigated, and the following features emerge from this studyg: the cobaltic ion is bound in the plane of the metal ion to phenanthroline and to the fl- and 7-phosphate groups; the apical positions are occupied by the N-7 of the adenine ring and by the 0~- anion, which is an exchangeable ligand that can be replaced by a protein ligand. ' Y. Hochman, C. Carmeli, A. Lanir, and M. M. Werber, to be published (1977). '~It has been established that Co-(phen)-ATP is not hydrolyzed by myosin or by coupling factor 1. This is probably due to the inertness of the complex, which precludes liberation of ligands in the medium. " A. Danchin and M. M. Werber, to be published (1977).
[32] T h e A c t i v e S i t e o f R i b o n u c l e o s i d e Diphosphate Reductase 1
By LARS THELANDER, JOHN HOBBS, and FRITZ ECKSTEIN Ribonucleotide reductase of Escherichia coli, which catalyzes the reduction of ribonucleoside 5'-diphosphates to 2"-deoxynucleoside 5'-diphosphates, consists of two nonidentical subunits, proteins B1 and B2. In the presence of Mg 2+, the two subunits form a 1:1 complex of active enzyme. 2 When separated, neither subunit has any known biological activity. Protein B1 has a molecular weight of 160,000, contains the active dithiols, is capable of interacting with thioredoxin, and contains 1L. Thelander, B. Larsson, J. Hobbs, and F. Eckstcin, J. Biol. Chem. 251, 1398 (1976). L. Thelander, J. Biol. Chem. 248, 4591 (1973).
322
ENZYMES, ANTIBODIES,. AND OTHER PROTEINS
[32]
binding sites both for the ribonucleoside diphosphate substrates ~ and for the nucleoside triphosphate effectors. 4 Protein B2 has a molecular weight of 78,000 and contains bound nonheme iron and an organic free radical essential for activity. 2,5 The radical gives rise to a characteristic light absorption at 410 nm. B2 is inactivated by removal of the iron or by destruction of the radical with hydroxylamine. No binding of substrates or effeetors to B2 can be demonstrated. Here, the inactivation of ribonucleotide reductase by 2t-deoxy-2 'chlorocytidine and by 2P-deoxy-2"-chlorouridine 5'-diphosphate, as well as by 2'-deoxy-2'-azidocytidine 5'-diphosphate, is described. 1 The results indicate that both B1 and B2 contribute to the active site of the enzyme and that the radical present in B2 directly participates in the catalytic process together with the redox active dithiols of B1.
Synthesis of Substrate Analogs 2P-Chloro-2'-deoxycytidine ~ 2'-Chloro-2'-deoxy-4-thiouridine 7 (278.5 mg; 1 mmole) is dissolved in water (200 ml), and a 2-ml aliquot of a solution containing 4.725 g of anhydous sodium sulfite and 1.188 g of sodium hyposulfite in 50 ml of water is added. A brisk stream of air is drawn through the solution via a sintered-glass disk. Additional 2-ml aliquots are added at hourly intervals up to 4 hr. Examination of the UV spectrum of the reaction solution shows ~,lax changing from 329 to 318 nm. After 5.5 hr, bubbling is stopped and 1 M ammonium acetate (10 ml) is added to the solution with sufficient concentrated ammonia to bring the pH to 8.7. The solution is stirred magnetically. After about 1 hr the pH is readjusted to 8.7 with a little more ammonia, and the solution is allowed to stir overnight. A large new maximum at about 270 nm is evident, the c2~o:~31sbeing about 50. The aqueous solution is evaporated, traces of water being removed by addition and evaporation of pyridine using an oil pump. The temperature throughout the reaction is not allowed to rise above 25 ° . The residue is thoroughly triturated with dry pyridine (3 X 15 ml), and the inorganic salts are removed by filtration. The pyridine is evaporated, and traces of U. V. DSbeln, J. Biol. Chem. 251, 3616 (1976). 4N. C. Brown and P. Reichard, J. Mol. Biol. 46, 39 (1969). C. L. Atkln, L. Thelander, P. Reichard, and G. Lang, J. Biol. Chem. 298, 7464 (1973). ~J. ttobbs, H. Sternbach, M. Sprinzl, and F. Eckstein, Biochemistry 11, 4336 (1972). 7I. L. Doerr and J. J. Fox, J. Org. Chem. 32, 1463 (1967).
[32]
RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE
323
pyridine are removed from the residual gum by addition and evaporation of water. The gum is dissolved in a little methanol and applied to thinlayer chromatography (TLC) plates (2 mm thickness), which are developed with methanol-chloroform (40:60, v/v). The maior band (Ry 0.74) is excised, and the product is eluted with methanol. The methanolic solution is evaporated to give a gum that does not crystallize, but yields a single spot on TLC (R~ 0.62) in the above system, and on paper chromatography (Ry 0.72) in system A. The yield may be estimated spectrophotometrically as 6.48 X 103 A2~9 units in H._,O (81%). Upon thawing a frozen concentrated aqueous solution of the product (about 31 mg/ml), white crystals are obtained. These show a melting phase, 109°-115 °, followed by formation of a new crystalline phase in the range 118°-130 °. This sinters at 190°-220 °, decomposing at 230°-240 °. (or.7) 2'-Azido-2'-deoxyuridine
2'-Azido-2'-deoxyuridine is prepared by a slight modification of a procedure described by Verheyden et al. s Uridine (10 g) and diphenylcarbonate (12 g) are stirred in hexamethylphosphotriamide (80 ml) in an oil bath at 140 °, and NaHC03 {0.24 g) is added. After cessation of bubbling, approximately 30 min, LiN3 (8 g) is added. After about 2 hr at 140 ° the solution is cooled, diluted with H..,O (16 ml), and extracted with CHC13 (2 }( 200 ml). The combined CHC13 extracts are extracted with H20 (2 }( 16 ml), and the combined aqueous solution is again extracted with CHC13 (3 X 200 ml). The CHCI.~ extracts from this last step are evaporated under reduced pressure. The residue is triturated with a mixture of acetone (160 ml) and MeOH (60 ml) and filtered, and the filtrate is evaporated. The remaining oil is chromatographed on SiO: (200 g) which has been equilibrated with acetone; the column is eluted with acetone. Fractions containing the product are combined and further purified by preparative TLC (6 plates, 20 X 40 cm, 2-ram layer of Si02). The plates are developed with acetone/ethyl acetate (1:1). The product containing bands were scraped out and eluted with acetone. The acetone solution is evaporated, and the residue is taken up in pyridine (45 ml) to remove Si02 and filtered; the filtrate is evaporated and reevaporated several times with H~O to remove traces of pyridine. The remaining slightly yellow oil is homogeneous by TLC (acetone/ethyl acetate, 1:1) with a yield of 50%. This material may be used without further purification for the synthesis of 2"-azido-2'-deoxycytidine. On standing at room temperature, the oil crystallizes. J. P. H. Verheyden, D. Wagner, and J. G. Moffatt, J. Org. Chem. 36, 250 (1971).
324
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[32]
To remove the yellow color, the material (approximately 2 mmoles) may be applied to a Dowex 1 X 4 ion-exchange column (1.7 X 17 cm, OH- form) and the column is washed sequentially with H20 (500 ml) and with 50% aqueous MeOH (500 ml); the compound is eluted with 0.1 M triethylammonium bicarbonate. The eluate is evaporated, and the buffer is removed by repeated evaporations with MeOH. The residue is triturated with acetone (20 ml) and filtered; the acetone solution is evaporated, the residue is applied to a SiO~ column (1.7 X 17 cm), and the column is eluted with acetone. The acetone solution is evaporated; the residue crystallizes on standing: white needles, m.p. 139°-147 °, with darkening and decomposition which became very rapid above 180 ° . Yield 95%.
2P-Azido-2'-deoxycytidine °,1° 2P-Azido-2'-deoxy-3',5'-diacetyluridine s (1.0 g) is dissolved in 13.9 ml of ethanol-free chloroform, and dry dimethylformamide (0.139 ml) and thionyl chloride (2.2 ml) are added. The solution is heated under reflux for 6.5 hr, cooled to room temperature, evaporated, and dissolved in methanol (60 ml) that is 50% saturated with ammonia. The solution is stirred at room temperature for 5 days. TLC on SiO~ (methanolchloroform, 40:60, v/v) shows the major product to have Rs 0.62. The solution is evaporated, and the product is separated by preparative TLC in the above system; the required band elutes with methanol. The eluate is evaporated and the residue is dissolved in water and applied to a column of Dowex 1 X 2 (OH-) (1.7 X 21.5 cm). The column is washed with water, and the required product is eluted with methanol-water (30: 70, v/v). The solvent is evaporated; the remaining gum, dissolved in a little ethanol, yields white crystals when stored at 5 ° . The yield (determined spectrophotometrically) is 47%, m.p. 215 ° (decomposition). Phosphorylation of Nucleosides
2'-C hloro-2P-deoxyeytidine 5'-Phosphate; 2'-C hloro-2"-deoxycytidine 5"-Monophosphate 2'-Chloro-2'-deoxycytidine (0.5 mmole) is dissolved in triethylphosphate (2 ml) and cooled to 0°; redistilled phosphoryl chloride (0.3 ml), also cooled to the same temperature, is added. The reaction mixture is 9j. ttobbs and F. Eckstein, in "Nucleic Acid Chemistry, New and/or Improved Synthetic Procedures" (L. B. Townsend, ed.), Vol. I, in press. Wiley, New York, 1977. loj. Hobbs, H. Sternbach, M. Sprinzl, and F. Eckstein, Biochemistry 12, 5138 (1973).
[32]
RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE
325
maintained at ice temperature. After 80 min, when the reaction appears to be far advanced as iudged by TLC (on SiO2 plates, developing with methanol/chloroform, 1:1, v/v), the reaction flask is placed on a vacuum rotary evaporator for 15 min to remove excess phosphoryl chloride, and then cooled to 0 ° with a small piece of ice added. After 15 min, the solution is again evaporated to remove traces of water and subsequently cooled to 0 °, and triethylamine (2.5 ml) is added. A granular precipitate forms at once. On addition of a couple of drops of water, the precipitate becomes gummy, and the solution is decanted and discarded. The gum is dissolved in a little water and applied to a column of DEAE-cellulose (32 }( 2.1 cm), which is washed thoroughly with water and then eluted with a linear gradient of triethylammonium bicarbonate (0-0.15 M in 3 liters). The major UV-absorbing peak is collected and evaporated; traces of triethylamine are removed by addition of methanol and evaporation, to obtain 2'-chloro-2'-deoxycytidine 5'-monophosphate (3150 A~o units, 79%) homogeneous on electrophoresis at pH 7.5 (30 volts/cm, 90 min). 2'-Azido-2'-deoxycytidine 5'-phosphate is prepared in the same manner. Preparation of 5'-diphosphates from the nucleoside 5'-phosphates was carried out according to Michelson. n Protein Inactivation
Inactivation o] Protein B1 by 2-Deoxy-2'-chlorocytidi~e 5"-Diphosphate (CclDP) Addition of CclDP to a solution containing ribonucleoside diphosphate reductase resulted in inactivation of the B1 subunit. When increasing amounts of CclDP were incubated with proteins B1 and B2 in the presence of Mg -°* and a positive effector, there was progressive inactiva~ tion of B1; complete inactivation was attained with 4 moles of CclDP per mole of B1. Inactivation of B1 showed an absolute requirement for active protein B2. Protection of B1 against inactivation by CclDP was observed when increasing amounts of CDP were added to B1 assay mixtures together with a fixed amount of CclDP. The inactivation of B1 was much faster in the presence of a positive effector (ATP or dTTP) than in the presence of a negative one (dATP or dGTP). As reaction products of the inactivation, free base (cytosine), chloride ion, and a compound which behaved electrophorctically and ehromatographically like 2-deoxy-5-diphosphate could be identified. As a result of the reaction with CclDP a loss of titratable SH-group in protein B1 was observed. Addition of an excess of CelDP to a B1-B2 mixture resulted " A. M. Michelson, Biochim. Biophys. Acta 91, 1 (19~4).
326
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[32]
in the loss of about 6 titratable SH groups per mole of B1 at complete inactivation. A similar loss of titratable sulfhydryls occurred on addition of the normal substrate CDP but without inactivation. 1~ In the latter case the SH groups were easily regenerated by addition of dithiothreitol, whereas neither sulfhydryl nor B1 activity could be regenerated after inactivation by CclDP, even in the presence of 6 M guanidinium hydrochloride. The nature of the sulfhydryl modification remains unknown. When CclDP was added to a B1-B2 mixture a slow increase in absorbance at 320 nm was observed, which was removed with B1 after chromatography on dATP-Sepharose. -~ This modification is tentatively believed to be one of tryptophan.
Inactivation of Protein B2 by 2"-Deoxy-2'-azidocytidine 5'-Diphosphate ( CzDP) Addition of increasing amounts of CzDP to B1-B2 mixtures gave a progressive decrease in B2 activity until 100% inactivation was attained when approximately 0.2 mole of CzDP had been added per mole of B2. In the absence of B1 no inactivation was observed. Only B1 with redox active dithiols could fulfill this function. This indicated that reduction of CzDP was required for inactivation of B2. Increasing amounts of CDP together with a constant amount of CzDP resulted in a decreased inactivation of B2 indicating competition between the analog and substrate for the same site on the reductase. The reductase had to be in an active conformation in order to be inactivated by CzDP. Addition of the negative effector dATP slowed down the inactivation while further addition of ATP increased it. When the B1-B2 complex was incubated with an excess of [fl_3~p] CzDP, analyses of the reaction mixture after complete inactivation showed the loss of 2.3 moles of sulfhydryls in B1. Chromatography on Sephadex showed that no radioactivity was bound to protein. The characteristic 410 nm absorbance peak of protein B2 ~ had been lost completely after inactivation. Iron analyses showed that this was not due to loss of iron. Removal of iron by dialysis and readdition of Fe ~÷ resulted in a regaining of activity and the reappearance of the absorption at 420 rim. It is concluded that inactivation of B2 by CzDP is due to the selective destruction of the free radical necessary for B2 activity. Comments
Both the 2'-chloro- and the 2'-azido-2'-deoxyribonucleoside diphosphates are bound to the substrate binding site of the BI-B2 complex as ~"L. Thelander, I. Biol. Chem. 249, 4858 (1974).
[33]
ADENOSINE DEAMINASE
327
shown by the influence of allosteric effectors on the inactivation, protection of the enzyme by substrate, the inability of the monophosphates to act as inhibitors, and the requirement for the presence of both B1 and B2 to achieve inactivation. Neither CclDP nor CzDP reacted with protein B1 or B2 alone or with dithiothreitol. We suggest that the substrate analogs were converted to reactive species that, in turn, inactivated either B1 or B2 by modification of functional groups of the active site participating in the catalytic process. In B1 the redox active dithiols were modified and in B2 the radical was destroyed. In this activity, the chloro and azido derivatives behaved as irreversible enzyme inhibitors, i.e., kcat inhibitors. These are characterized as unreactive compounds (proinhibitors) that are converted to a highly reactive form (inhibitor) within an active site by the specific action of a particular enzyme.13 CzDP is a very effective inhibitor, since it inactivated protein B2 stoichiometrieally. The value of about 0.2 mole of CzDP required for total inactivation of 1 mole of B2 agrees with the known variable and low content of free radical in the B2 preparations. 1. CclDP is somewhat less effective, providing about 50% inactivation of B1 on addition of stoichiometric amounts of chloro derivative (2 moles of CcIDP per mole of B1 based on two binding sites for ribonucleoside diphosphates per mole of B13). Earlier data ~ and those derived from these inhibition studies can be summarized in a model of ribonucleoside diphosphate reductase in which the active site is formed both from B1 and B2. It contains active dithiols contributed by B1 and a free radical contributed by B2. The active dithiols donate the electrons required for ribonucleotide reduction while participating in catalysis; the function and nature of the free radical remain unknown. ~' R. R. Rando, Science 185, 320 (1974). See also this volume [3] and [12]. 14A. Ehrenberg and P. Reichard, J. Biol. Chem. 247, 3485 (1972). 1~N. C. Brown, Z. M. Canellakis, B. Lundin, P. :Reichard, and L. Thelander, Ew'. J. Biochem. 9, 561 (1969).
[33] A d e n o s i n e D e a m i n a s e
By
GIOVANNI RONCA, ANTONIO LUCACCHINI,and CARLO ALFONSO ROSSI
Adenosine deaminase plays a key role in adenosine metabolism. This nueleoside has some important pharmacological and toxic effects. An adenosine deaminase deficiency observed in some cases of severe con-
R I B O N U C L E O S I DDIPHOSPHATE E REDUCTASE
321
sary when long (12 hr) incubations are employed. For example, at pH 7 and 0 °, the half-life of Co-(phen)-ATP was found to be 20 hr. The stoichiometry obtained both in the case of myosin and its subfragments 3,* and in the case of coupling factor 14,7 indicates that the labeling occurs only at specific sites on the enzymes. Moreover, the fact that it is possible to separate the labeled enzymes from excess reagent is by itself a proof that the association with the complex is irreversible. Co-(phen)-ATP was also shown to behave as a competitive inhibitor of myosin a and of coupling factor 1 when added directly into the assay medium. 8 Moreover, in the case of heavy meromyosin, both Mg ATP and Mg ADP could protect against labeling by Co-(phen)-ATP. * Experiments with thiol reagents (p-hydroxymercuribenzoate and N-ethylmaleimide) seem to indicate that Co-(phen)-ATP either binds to or protects some essential SH groups in the active site of myosin. The structure of Co-(phen)-ATP has been investigated, and the following features emerge from this studyg: the cobaltic ion is bound in the plane of the metal ion to phenanthroline and to the fl- and 7-phosphate groups; the apical positions are occupied by the N-7 of the adenine ring and by the 0~- anion, which is an exchangeable ligand that can be replaced by a protein ligand. ' Y. Hochman, C. Carmeli, A. Lanir, and M. M. Werber, to be published (1977). '~It has been established that Co-(phen)-ATP is not hydrolyzed by myosin or by coupling factor 1. This is probably due to the inertness of the complex, which precludes liberation of ligands in the medium. " A. Danchin and M. M. Werber, to be published (1977).
[32] T h e A c t i v e S i t e o f R i b o n u c l e o s i d e Diphosphate Reductase 1
By LARS THELANDER, JOHN HOBBS, and FRITZ ECKSTEIN Ribonucleotide reductase of Escherichia coli, which catalyzes the reduction of ribonucleoside 5'-diphosphates to 2"-deoxynucleoside 5'-diphosphates, consists of two nonidentical subunits, proteins B1 and B2. In the presence of Mg 2+, the two subunits form a 1:1 complex of active enzyme. 2 When separated, neither subunit has any known biological activity. Protein B1 has a molecular weight of 160,000, contains the active dithiols, is capable of interacting with thioredoxin, and contains 1L. Thelander, B. Larsson, J. Hobbs, and F. Eckstcin, J. Biol. Chem. 251, 1398 (1976). L. Thelander, J. Biol. Chem. 248, 4591 (1973).
322
ENZYMES, ANTIBODIES,. AND OTHER PROTEINS
[32]
binding sites both for the ribonucleoside diphosphate substrates ~ and for the nucleoside triphosphate effectors. 4 Protein B2 has a molecular weight of 78,000 and contains bound nonheme iron and an organic free radical essential for activity. 2,5 The radical gives rise to a characteristic light absorption at 410 nm. B2 is inactivated by removal of the iron or by destruction of the radical with hydroxylamine. No binding of substrates or effeetors to B2 can be demonstrated. Here, the inactivation of ribonucleotide reductase by 2t-deoxy-2 'chlorocytidine and by 2P-deoxy-2"-chlorouridine 5'-diphosphate, as well as by 2'-deoxy-2'-azidocytidine 5'-diphosphate, is described. 1 The results indicate that both B1 and B2 contribute to the active site of the enzyme and that the radical present in B2 directly participates in the catalytic process together with the redox active dithiols of B1.
Synthesis of Substrate Analogs 2P-Chloro-2'-deoxycytidine ~ 2'-Chloro-2'-deoxy-4-thiouridine 7 (278.5 mg; 1 mmole) is dissolved in water (200 ml), and a 2-ml aliquot of a solution containing 4.725 g of anhydous sodium sulfite and 1.188 g of sodium hyposulfite in 50 ml of water is added. A brisk stream of air is drawn through the solution via a sintered-glass disk. Additional 2-ml aliquots are added at hourly intervals up to 4 hr. Examination of the UV spectrum of the reaction solution shows ~,lax changing from 329 to 318 nm. After 5.5 hr, bubbling is stopped and 1 M ammonium acetate (10 ml) is added to the solution with sufficient concentrated ammonia to bring the pH to 8.7. The solution is stirred magnetically. After about 1 hr the pH is readjusted to 8.7 with a little more ammonia, and the solution is allowed to stir overnight. A large new maximum at about 270 nm is evident, the c2~o:~31sbeing about 50. The aqueous solution is evaporated, traces of water being removed by addition and evaporation of pyridine using an oil pump. The temperature throughout the reaction is not allowed to rise above 25 ° . The residue is thoroughly triturated with dry pyridine (3 X 15 ml), and the inorganic salts are removed by filtration. The pyridine is evaporated, and traces of U. V. DSbeln, J. Biol. Chem. 251, 3616 (1976). 4N. C. Brown and P. Reichard, J. Mol. Biol. 46, 39 (1969). C. L. Atkln, L. Thelander, P. Reichard, and G. Lang, J. Biol. Chem. 298, 7464 (1973). ~J. ttobbs, H. Sternbach, M. Sprinzl, and F. Eckstein, Biochemistry 11, 4336 (1972). 7I. L. Doerr and J. J. Fox, J. Org. Chem. 32, 1463 (1967).
[32]
RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE
323
pyridine are removed from the residual gum by addition and evaporation of water. The gum is dissolved in a little methanol and applied to thinlayer chromatography (TLC) plates (2 mm thickness), which are developed with methanol-chloroform (40:60, v/v). The maior band (Ry 0.74) is excised, and the product is eluted with methanol. The methanolic solution is evaporated to give a gum that does not crystallize, but yields a single spot on TLC (R~ 0.62) in the above system, and on paper chromatography (Ry 0.72) in system A. The yield may be estimated spectrophotometrically as 6.48 X 103 A2~9 units in H._,O (81%). Upon thawing a frozen concentrated aqueous solution of the product (about 31 mg/ml), white crystals are obtained. These show a melting phase, 109°-115 °, followed by formation of a new crystalline phase in the range 118°-130 °. This sinters at 190°-220 °, decomposing at 230°-240 °. (or.7) 2'-Azido-2'-deoxyuridine
2'-Azido-2'-deoxyuridine is prepared by a slight modification of a procedure described by Verheyden et al. s Uridine (10 g) and diphenylcarbonate (12 g) are stirred in hexamethylphosphotriamide (80 ml) in an oil bath at 140 °, and NaHC03 {0.24 g) is added. After cessation of bubbling, approximately 30 min, LiN3 (8 g) is added. After about 2 hr at 140 ° the solution is cooled, diluted with H..,O (16 ml), and extracted with CHC13 (2 }( 200 ml). The combined CHC13 extracts are extracted with H20 (2 }( 16 ml), and the combined aqueous solution is again extracted with CHC13 (3 X 200 ml). The CHCI.~ extracts from this last step are evaporated under reduced pressure. The residue is triturated with a mixture of acetone (160 ml) and MeOH (60 ml) and filtered, and the filtrate is evaporated. The remaining oil is chromatographed on SiO: (200 g) which has been equilibrated with acetone; the column is eluted with acetone. Fractions containing the product are combined and further purified by preparative TLC (6 plates, 20 X 40 cm, 2-ram layer of Si02). The plates are developed with acetone/ethyl acetate (1:1). The product containing bands were scraped out and eluted with acetone. The acetone solution is evaporated, and the residue is taken up in pyridine (45 ml) to remove Si02 and filtered; the filtrate is evaporated and reevaporated several times with H~O to remove traces of pyridine. The remaining slightly yellow oil is homogeneous by TLC (acetone/ethyl acetate, 1:1) with a yield of 50%. This material may be used without further purification for the synthesis of 2"-azido-2'-deoxycytidine. On standing at room temperature, the oil crystallizes. J. P. H. Verheyden, D. Wagner, and J. G. Moffatt, J. Org. Chem. 36, 250 (1971).
324
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[32]
To remove the yellow color, the material (approximately 2 mmoles) may be applied to a Dowex 1 X 4 ion-exchange column (1.7 X 17 cm, OH- form) and the column is washed sequentially with H20 (500 ml) and with 50% aqueous MeOH (500 ml); the compound is eluted with 0.1 M triethylammonium bicarbonate. The eluate is evaporated, and the buffer is removed by repeated evaporations with MeOH. The residue is triturated with acetone (20 ml) and filtered; the acetone solution is evaporated, the residue is applied to a SiO~ column (1.7 X 17 cm), and the column is eluted with acetone. The acetone solution is evaporated; the residue crystallizes on standing: white needles, m.p. 139°-147 °, with darkening and decomposition which became very rapid above 180 ° . Yield 95%.
2P-Azido-2'-deoxycytidine °,1° 2P-Azido-2'-deoxy-3',5'-diacetyluridine s (1.0 g) is dissolved in 13.9 ml of ethanol-free chloroform, and dry dimethylformamide (0.139 ml) and thionyl chloride (2.2 ml) are added. The solution is heated under reflux for 6.5 hr, cooled to room temperature, evaporated, and dissolved in methanol (60 ml) that is 50% saturated with ammonia. The solution is stirred at room temperature for 5 days. TLC on SiO~ (methanolchloroform, 40:60, v/v) shows the major product to have Rs 0.62. The solution is evaporated, and the product is separated by preparative TLC in the above system; the required band elutes with methanol. The eluate is evaporated and the residue is dissolved in water and applied to a column of Dowex 1 X 2 (OH-) (1.7 X 21.5 cm). The column is washed with water, and the required product is eluted with methanol-water (30: 70, v/v). The solvent is evaporated; the remaining gum, dissolved in a little ethanol, yields white crystals when stored at 5 ° . The yield (determined spectrophotometrically) is 47%, m.p. 215 ° (decomposition). Phosphorylation of Nucleosides
2'-C hloro-2P-deoxyeytidine 5'-Phosphate; 2'-C hloro-2"-deoxycytidine 5"-Monophosphate 2'-Chloro-2'-deoxycytidine (0.5 mmole) is dissolved in triethylphosphate (2 ml) and cooled to 0°; redistilled phosphoryl chloride (0.3 ml), also cooled to the same temperature, is added. The reaction mixture is 9j. ttobbs and F. Eckstein, in "Nucleic Acid Chemistry, New and/or Improved Synthetic Procedures" (L. B. Townsend, ed.), Vol. I, in press. Wiley, New York, 1977. loj. Hobbs, H. Sternbach, M. Sprinzl, and F. Eckstein, Biochemistry 12, 5138 (1973).
[32]
RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE
325
maintained at ice temperature. After 80 min, when the reaction appears to be far advanced as iudged by TLC (on SiO2 plates, developing with methanol/chloroform, 1:1, v/v), the reaction flask is placed on a vacuum rotary evaporator for 15 min to remove excess phosphoryl chloride, and then cooled to 0 ° with a small piece of ice added. After 15 min, the solution is again evaporated to remove traces of water and subsequently cooled to 0 °, and triethylamine (2.5 ml) is added. A granular precipitate forms at once. On addition of a couple of drops of water, the precipitate becomes gummy, and the solution is decanted and discarded. The gum is dissolved in a little water and applied to a column of DEAE-cellulose (32 }( 2.1 cm), which is washed thoroughly with water and then eluted with a linear gradient of triethylammonium bicarbonate (0-0.15 M in 3 liters). The major UV-absorbing peak is collected and evaporated; traces of triethylamine are removed by addition of methanol and evaporation, to obtain 2'-chloro-2'-deoxycytidine 5'-monophosphate (3150 A~o units, 79%) homogeneous on electrophoresis at pH 7.5 (30 volts/cm, 90 min). 2'-Azido-2'-deoxycytidine 5'-phosphate is prepared in the same manner. Preparation of 5'-diphosphates from the nucleoside 5'-phosphates was carried out according to Michelson. n Protein Inactivation
Inactivation o] Protein B1 by 2-Deoxy-2'-chlorocytidi~e 5"-Diphosphate (CclDP) Addition of CclDP to a solution containing ribonucleoside diphosphate reductase resulted in inactivation of the B1 subunit. When increasing amounts of CclDP were incubated with proteins B1 and B2 in the presence of Mg -°* and a positive effector, there was progressive inactiva~ tion of B1; complete inactivation was attained with 4 moles of CclDP per mole of B1. Inactivation of B1 showed an absolute requirement for active protein B2. Protection of B1 against inactivation by CclDP was observed when increasing amounts of CDP were added to B1 assay mixtures together with a fixed amount of CclDP. The inactivation of B1 was much faster in the presence of a positive effector (ATP or dTTP) than in the presence of a negative one (dATP or dGTP). As reaction products of the inactivation, free base (cytosine), chloride ion, and a compound which behaved electrophorctically and ehromatographically like 2-deoxy-5-diphosphate could be identified. As a result of the reaction with CclDP a loss of titratable SH-group in protein B1 was observed. Addition of an excess of CelDP to a B1-B2 mixture resulted " A. M. Michelson, Biochim. Biophys. Acta 91, 1 (19~4).
326
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[32]
in the loss of about 6 titratable SH groups per mole of B1 at complete inactivation. A similar loss of titratable sulfhydryls occurred on addition of the normal substrate CDP but without inactivation. 1~ In the latter case the SH groups were easily regenerated by addition of dithiothreitol, whereas neither sulfhydryl nor B1 activity could be regenerated after inactivation by CclDP, even in the presence of 6 M guanidinium hydrochloride. The nature of the sulfhydryl modification remains unknown. When CclDP was added to a B1-B2 mixture a slow increase in absorbance at 320 nm was observed, which was removed with B1 after chromatography on dATP-Sepharose. -~ This modification is tentatively believed to be one of tryptophan.
Inactivation of Protein B2 by 2"-Deoxy-2'-azidocytidine 5'-Diphosphate ( CzDP) Addition of increasing amounts of CzDP to B1-B2 mixtures gave a progressive decrease in B2 activity until 100% inactivation was attained when approximately 0.2 mole of CzDP had been added per mole of B2. In the absence of B1 no inactivation was observed. Only B1 with redox active dithiols could fulfill this function. This indicated that reduction of CzDP was required for inactivation of B2. Increasing amounts of CDP together with a constant amount of CzDP resulted in a decreased inactivation of B2 indicating competition between the analog and substrate for the same site on the reductase. The reductase had to be in an active conformation in order to be inactivated by CzDP. Addition of the negative effector dATP slowed down the inactivation while further addition of ATP increased it. When the B1-B2 complex was incubated with an excess of [fl_3~p] CzDP, analyses of the reaction mixture after complete inactivation showed the loss of 2.3 moles of sulfhydryls in B1. Chromatography on Sephadex showed that no radioactivity was bound to protein. The characteristic 410 nm absorbance peak of protein B2 ~ had been lost completely after inactivation. Iron analyses showed that this was not due to loss of iron. Removal of iron by dialysis and readdition of Fe ~÷ resulted in a regaining of activity and the reappearance of the absorption at 420 rim. It is concluded that inactivation of B2 by CzDP is due to the selective destruction of the free radical necessary for B2 activity. Comments
Both the 2'-chloro- and the 2'-azido-2'-deoxyribonucleoside diphosphates are bound to the substrate binding site of the BI-B2 complex as ~"L. Thelander, I. Biol. Chem. 249, 4858 (1974).
[33]
ADENOSINE DEAMINASE
327
shown by the influence of allosteric effectors on the inactivation, protection of the enzyme by substrate, the inability of the monophosphates to act as inhibitors, and the requirement for the presence of both B1 and B2 to achieve inactivation. Neither CclDP nor CzDP reacted with protein B1 or B2 alone or with dithiothreitol. We suggest that the substrate analogs were converted to reactive species that, in turn, inactivated either B1 or B2 by modification of functional groups of the active site participating in the catalytic process. In B1 the redox active dithiols were modified and in B2 the radical was destroyed. In this activity, the chloro and azido derivatives behaved as irreversible enzyme inhibitors, i.e., kcat inhibitors. These are characterized as unreactive compounds (proinhibitors) that are converted to a highly reactive form (inhibitor) within an active site by the specific action of a particular enzyme.13 CzDP is a very effective inhibitor, since it inactivated protein B2 stoichiometrieally. The value of about 0.2 mole of CzDP required for total inactivation of 1 mole of B2 agrees with the known variable and low content of free radical in the B2 preparations. 1. CclDP is somewhat less effective, providing about 50% inactivation of B1 on addition of stoichiometric amounts of chloro derivative (2 moles of CcIDP per mole of B1 based on two binding sites for ribonucleoside diphosphates per mole of B13). Earlier data ~ and those derived from these inhibition studies can be summarized in a model of ribonucleoside diphosphate reductase in which the active site is formed both from B1 and B2. It contains active dithiols contributed by B1 and a free radical contributed by B2. The active dithiols donate the electrons required for ribonucleotide reduction while participating in catalysis; the function and nature of the free radical remain unknown. ~' R. R. Rando, Science 185, 320 (1974). See also this volume [3] and [12]. 14A. Ehrenberg and P. Reichard, J. Biol. Chem. 247, 3485 (1972). 1~N. C. Brown, Z. M. Canellakis, B. Lundin, P. :Reichard, and L. Thelander, Ew'. J. Biochem. 9, 561 (1969).
[33] A d e n o s i n e D e a m i n a s e
By
GIOVANNI RONCA, ANTONIO LUCACCHINI,and CARLO ALFONSO ROSSI
Adenosine deaminase plays a key role in adenosine metabolism. This nueleoside has some important pharmacological and toxic effects. An adenosine deaminase deficiency observed in some cases of severe con-
