[16] Pyrimidine nucleoside and deoxynucleoside phosphorylases

118

NUCLEIC ACID COMPONENTS

[16]

pentose phosphates in presence of hypoxanthine, xanthine, adenine, 2s guanine, 8-azaguanine, 6-mercaptopurine, 6-methylpurine, and 2,6-diaminopurine indicated that the corresponding nucleosides were formed. Purine, isoguanine, 8-azaxanthine, 8-chloroxanthine, 4-amino-5-imidazole carboxamide, uric acid, C-2-substituted purines, cytosine, and thymine were not utilized. The equilibrium was about 85% in favor of nucleoside formation with hypoxanthine and R1-P or dR1-P as substrates. Both dR1-P and R1-P were obtained in comparatively good yields with either purified or crude enzyme preparations using guanosine and orthophosphate as substrafes. 11 With guanosine or deoxyguanosine as substrates the equilibrium tended to favor pentose phosphate formation, presumably because of the insolubility of the guanine and the fact that much of this was deaminated to xanthine by a guanine deaminase 29 present in the muscle. Guanine, but not xanthine, has been said to inhibit nucleoside phosphorylases2 No phosphatase activity was detected in the preparations, but both crude and purified preparations possessed phosphoribomutase activity which reduced the yield of R1-P from guanosine due to R5-P formation. The optimum pH was 7.0-7.5. H. L. A. Tarr and A. G. Comer, Can. J. Biochem. 42, 1527 (1964) . ~J. Roy, Can. J. Biochem. 44, 1093 (1966).

[16] Pyrimidine Nucleoside and Deoxynucleoside Phosphorylases B y W. E. RAZZELL

Preparations of pyrimidine-specific phosphorylases which are also specific for deoxyribose, ribose, or either one, may be found useful in particular circumstances for the characterization or preparation of a variety of N-pentosides. The purification, assay, and properties of two enzymes are described: pyrimidine deoxynucleoside phosphorylase (socalled thymidine phosphorylase, EC 2.4.2.4. thymidine: orthophosphate deoxyribosyltransferase) fl'om Escherichia colt; and uridine phosphorylase (EC 2.4.2.3 uridine: orthophosphate ribosyltransferase) from Escherichia colt. Uridine (thymidine) phosphorylase from Ehrlich ascites cells (grown in mice) has been documented already in this series; 1 it is not specific with regard to the pentose moiety. 1p. Reichard and O. Skiild, Vol. VI [21].

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119

Thymidine Phosphorylase from E. coli TdR + P~~ T 4- dR1 - P

Assay Method Arsenolysis of ThymidineY ,s The replacement of inorganic phosphate by arsenate in the reaction mixture prevents an equilibrium condition and permits thymine detection when samples are transferred at intervals to tubes containing dilute alkali. Thus, to a l0 )< 75 mm test tube are added 0.20 ml of 32 mM thymidine in buffer (0.10 M sodium arsenate adjusted to pH 6.0 with 0.10M acetic acid) and water sufficient to bring the volume to 0.40 ml after the addition of enzyme. The tube is warmed to 45 °, enzyme is added, and samples of 50 ~l are removed at intervals to test tubes or silica euvettes containing 0.95 ml of 0.3N NaOH. The absorbance of the samples is determined at 300 m~, where 1 micromole of thymine liberation is represented by 3.61 O.D. units. Zero time values for reactions performed with purified enzyme or extracts of induced cells are less than 0.10 0.D., and linear rates are obtained up to 0.50 O.D. In very crude preparations, samples of 50 ~l, removed from the reaction mixture, may be allowed to react in the diphenylamine test for deoxyribose.4 Thyminolysis o] Deoxyribose 1-Phospkate. The inorganic phosphate liberated in the reaction of thymine or other base with deoxyribose 1phosphate is precipitated, washed, and determined, as follows. In a final volume of 0.20 ml are placed 0.75 micromole of pyrimidine base, 0.75 mieromole of deoxyribose 1-phosphate, 10 micromoles of Tris-HC1, pH 7.4, and water; the reaction mixture is warmed to 45 °, enzyme is added, and aliquots of 50 ~l are removed at intervals to micro centrifuge tubes immersed in crushed ice. (Three points are obtainable.) The centrifuge tubes contain 0.05 ml of a mixture consisting of 0.025 ml concentrated NH40H (30% NH3), 0.0125 ml of 0.25 M MgC12, and 0.0125 ml of 5 M NH4C12 After being chilled for 4 hours (or overnight in the refrigerator), the tubes are centrifuged, the supernatant solution is carefully aspirated, and the precipitate of MgNH,PO~ is resuspended in 0.10 ml of cold salt mixture (twice the volumes of NH4OH, MgC12, and NH4C1 given above). The suspension is chilled in ice 2 hours, the precipitate is centrifuged down once more and then dissolved in 0.20 ml of N HC1. Samples are removed for determination of P~ by the Chen procedure2 2M. Friedkin and D. Roberts, J. Biol. Chem. 207, 205 (1954). 3W. E. Razzell and H. G. Khorana, Biochim. Biophys. Acta 28, 562 (1958). 4W. E. Razzell and P. Casshyap, J. Biol. Chem. 239, 1789 (1964). 5The procedure previously given for this step3 contains a typographical omission: cone. NH~OH is crucial.

120

NUCLEIC ACID COMPONENTS

[16]

Definition o/Unit and Specific Activity. In all cases, a unit of enzyme is 1/60 of an International Unit, i.e., that amount which causes the conversion or the formation of 1 mieromole of pyrimidine base per hour; and specific activity is units per milligram protein. Purification Procedure

Growth of Cells and Preparation of Extracts. Any strain of E. coli is suitable, with due regard for auxotrophic requirements, except thyminedependent mutants, which are not inducible. 4 Cultures in broth are inoculated into tubes of inorganic salts-glucose medium ~ and aerated overnight at 37 ° . The entire contents of the tubes (one or several) are transferred to 1 liter of the same medium and aerated at 37 ° until the absorbance at 650 m~ reaches 2.5, then the liter of culture is transferred to 14 liters of the same medium and aerated at 37 ° until the absorbance at 650 m~ reaches 0.50. Now, thymidine (unsterile) is added to a concentration of 0.5 mg/ml (i.e., 7.5 g in 15 liters) and growth of cells is continued until the end of the log phase (usually A65o -----2.5). The culture is chilled in ice, cells are recovered by centrifugation, washed with cold 0.01 M 2-mercaptoethanol (or neutral cysteine, GSH, etc.) to a density of about 8 X 101° per milliliter (A65o ----80). The suspension is exposed to sonic oscillation (10 minutes ab 4 ° in a 250-watt Raytheon 10 ke machine tuned to maximum power output), and debris is removed by centrifugation at 15,000 g for 10 minutes. The cell-free supernatant retains essentially full activity during storage at --18 °. Isoelectric and Protamine Precipitations. s The solution from the above step is stirred in the cold and adjusted to pH 5.25 with glacial acetic acid, care being taken not to lower the pH below 5.2 at any time. The turbid suspension is immediately centrifuged at 15,000 g for 10 minutes. The supernatant is adjusted as before, to pH 4.90, stirred for 5

~B. N. Ames and D. T. Dubin, J. Biol. Chem. 235, 769 (1960). The samples are brought to a volume of 0.30 ml in carefully washed test tubes, and to each is added 0.70 ml of reagent (1 volume of 10% ascorbic acid plus 6 volumes of a solution containing I0 ml 5 N H~SO4, 50 ml of 2.5% (NH4)6 MO702"6 H~O in 5 N HzSO~, and 240 ml of water). After 20 minutes at 45 °, the contents are transferred to cuvettes of 1-ml capacity and the absorbance is determined at 820 m#. A standard curve for P~ yields 0.25 ± 0.01 O.D. unit per 0.010 micromole). TMost inorganic salts media are suitable; e.g., I. Lieberman, Vol. VI [11]. However, we routinely use the following, in the ratio of 2:1:1 to avoid caramelization and precipitates: A, !A% K~HPO, plus 0.4% KH-.PO,:B, 0.04% MgSO4-7 H20 plus 0A% (NI~)~SO4:C, 1.6% glucose. The solutions A, B, C are sterilized and mixed when cool. 8 W. J. Peters and W. E. Razzell, unpublished results (1966).

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minutes, and centrifuged. The supernatant is adjusted to pH 6.8 with 2 N NH40H, and stirred in ice during the addition of 0.1 volume of protamine sulfate solution (20 mg/ml Eli Lilly protamine sulfate, 9 adjusted to pH 5.0 with N H2S04 and maintained in solution at 40°). The copious white precipitate is removed by centrifugation, and the supernatant fluid is stored at 4 ° (not --18 °) in a full, stoppered, container. At this stage, the preparation is suitable for most purposes and following dialysis overnight at 4 °, against two changes of 100 volumes of 0.005 M Tris-ttC1, pH 7.4, containing 0.010 M 2-mercaptoethanol, may be stored indefinitely at 4 ° if a few crystals of thymol are added. The specific activity is 450, recovery about 85%. Chromatography on Dowex 1. The undialyzed protamine supernatant is applied in the cold to the top of a column of Dowex l-X2, acetate form, equilibrated with 0.02 M sodium acetate-0.02 M 2-mercaptoethanol, pH 6.5. Fifty milliliters of enzyme may be put onto a column 3 cm diameter and 30 cm high. Fifty milliliters of equilibrating buffer is applied to the column, followed by 150 ml of 0.10M sodium acetate0.01 M 2-mercaptoethanol, pH 5.5. The peak of enzyme emerges ahead of the protein peak and is collected in 5-ml fractions. The specific activity is above 3500 in the leading half of the enzyme peak, and recovery is about 50%. The enzyme should be kept in a closed container with thymol added, at 4 ° . Properties Specificity. The enzyme is specific for fl-thymidine, and other pyrimidine deoxynucleosides most probably must be in the fl configuration.4 The specificity for deoxyribose 1-a-phosphate appears to be absolute, although reactions involving 3% or less conversion of other pentose phosphates or pentosides cannot yet be excluded, and numerous experiments with crude extracts of E. coli indicate some kind of enzymatic activities with several pentosides. 1° The uridine phosphorylase activity of the cell extract, which in induced cells is about 8% of the thymidine phosphorylase, is removed by isoelectric precipitation and is also very unstable; thus, the reaction of thymine or uracil with ribose 1-phosphate is less than 0.5%, in rate or extent, of that with equal concentrations of deoxyribose 1-phosphate2 Less than 5% of normal rate or extent of reaction is observed with pentose phosphates and pyrimidine bases shown in the table. Other types of protamine sulfate may give good results, but the Eli Lilly preparations are consistently satisfactory. Attempts to dispense with the protamine step result in highly unstable enzyme preparations3 I°H. Tono and S. S. Cohen, J. Biol. Chem. 237, 1271 (1962). These authors attribute the cleavage of arabinasyl-uracil to the activity of uridine phosphorylase.

122

NUCLEIC ACID COMPONENTS

[15]

SUBSTRATE SPECIFICITYOF Escherichia coli THYMIDINE PHOSPHORYLASE A. Pentose phosphates a Ribose-l-P D-Arabinofuranose-l-P L-Arabinofuranose-l-P D-Arabinopyranose-1 -P D-Ribopyranose-l-P D-Ribofuranose-l-~-P 2-Deoxy-D-ribofuranosyl-l-f~-P B. Pyrimidine bases ~

Micromoles/ml

Rate relative to deoxyribose-l-P

3.75 3.75 3.75 3.75 7.5 7.5 3.75, 7.5

0.005 b'¢ 0.06 b 0.06 b 0.03 ~ 0.03 b 0.03 ~ 0. 002C

Micromoles/ml

Rate relative to thymine

Uracil 5~Aminouracil 2-Thiothymine 2-Thiouracil 5-Fluorouracil 5-Bromouracil 5-Iodouracil

3.75 3.75 3.75 3.75 3.75 3.75 3.75, 7.5

0.70 c 0.32 b'~ 0.19 ~

C. Thymidine analogs

Micromoles/ml

Rate relative to thymidine

2',3'-Dideoxythymidine 3'-Iodothymidine 3'- or 5'-Acetylthymidine Thymidine 5'-carboxylate Thymidine isoester Xylofuranosyl thymine

16, 4 8 16, 4 4 8 8

O. 98b'~ 1.15b'c 0.69 b O. 74b

0.001 c 0.005 0.001 c 0.005 c 0.002 ° 0.01 b

a Pyrimidine base = thymine. b Enzyme from uninduced cells [W. E. Razzell and H. G. Khorana, Biochim. Biophys. Acta 28, 562 (1958)]. c Enzyme from induced cells [W. E. Razzell and P. Casshyap, J. Biol. Chem. 9.39, 1789 (1964)]. The following compounds at 3.75 micromoles/ml yielded rates of P~ liberation <5% that with thymine: 5-nitro-, 5-hydroxymethyl-, 5-ribosyl-, 5-thio- and 5,6-dimethyl-uracil; 2-aminouracil; 2-amino-, 4-dithio-, 2,4-dichloro-, and dihydrothymine; 2,4-dithiopyrimidine; cytosine; 5-methylcytosinei 6-methyl-, 5,6-dimethyl-, and 6-azauracil; 6-azathymine. In addition, 3-N-methylthymidine was not phosphorolyzed under standard assay conditions (16 micromoles/ml). J In the presence of deoxyribose 1-phosphate. T h e overall conclusion which is d r a w n from the specificity studies is, t h a t e v e r y position of the base a n d sugar of t h y m i d i n e is recognized b y the e n z y m e ; thus, it is impossible to p r e d i c t w h e t h e r other a n a l o g s m a y f u n c t i o n as s u b s t r a t e s or i n h i b i t o r s , b u t no p a r t of the molecule is of no i m p o r t a n c e to the enzyme.

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General Properties. The enzyme is dependent on a reducing agent for stability. It is inactivated rapidly at pH values below 4 and above 9; heating at 70 ° for 5 minutes results in complete loss of activity (in contrast with thymidine kinase11). Phosphorolysis and arsenolysis proceed best at pH 6.0 although thymine and a few bases which are substrates ~ (including 5-ethyluraciP 2) inhibit to some degree; whereas synthesis from deoxyribose 1-phosphate proceeds best at pH 7.4-7.6, and inhibition by substrate excess (thymine, etc.) is also observed. Uridine Phosphorylase from E. coli UR -4- P& ~ U -4- R1 - P~Assay Methods

Arsenolysis of uridine and the detection of uracil may be performed in a manner similar to the assay for thymidine arsenolysis, or ribose formation may be assessed by the orcinol reaction, is Thus, a reaction mixture of 0.40 ml is prepared, containing 0.050M sodium arsenateacetate buffer, pH 7.5, and 0.0025 M uridine, and warmed to 37 °. Following addition of enzyme, aliquots of 50 ~1 are transferred at intervals to tubes containing 0.95 ml of 0.30 N NaOH, and the absorbanee is determined at 290 m~. The formation of 1 micromole of uracil results in an increase in absorbance of 5.41 O.D. units. Alternatively, the aliquots may be transferred to tubes containing 0.95 ml of orcinol reagent and water, then heated after all aliquots have accumulated. Unfortunately, the uridine phosphorylase activity is not as stable as the thymidine phosphorylase; the increment of products with time is nonlinear and assay precision is low, even with crude extracts. Neither different assay pH, addition of reducing agents, nor replacement of arsenate by phosphate alleviate the problem. 14 Purification Procedure ~4,15

Growth of Cells and Preparation o] Extracts. No induction of uridine phosphorylase has been observed above normal levels found in several strains of E. coli? Therefore, cells may be grown as described above for thymidine phosphorylase, but without thymidine addition, and extracts prepared in the same manner. The uridine phosphorylase in the cell-free extract is stable during several weeks storage at 4 ° or during 1, R. Okazaki and A. Kornberg, J. Biol. Chem. ~39, 269 (1964). ~2M. Piechowska and D. Shugar, Biochim. Biophys. Res. Commun. 20, 768 (1965). i~ G. Ashwell, Vol. I I I [12].

14W. E. Razzell, unpublished results (1966). 1~L. M. Paege and F. Schlenk, Arch. Biochem. Biophys. 4@, 42 (1952).

124

NUCLEIC ACID COMPONENTS

[16]

the course of freezing and thawing. 14 Careful pH adjustment, temperature control, and timing of exposure to low pH during the precipitation step of the purification procedure for thymidine phosphorylase frequently permits good yields of uridine phosphorylase in the precipitated fraction. If the precipitate is quickly resuspended in 0.05 M phosphate containing 0.01 M 2-mercaptoethanol, pH 7.0, it may be used for subsequent steps in uridine phosphorylase purification, in place of the cell-free extract itself. Protamine Sul]ate and Ammonium Sulfate Precipitations. The cellfree extract (or redissolved precipitate therefrom) is stirred in ice during the addition of 0.1 volume of protamine sulfate solution (20 mg/ml Eli Lilly protamine sulfate, pH 5.0), 9 and the precipitate is removed by centrifugation at 15,000 g for 10 minutes in the cold. The supernatant is stirred in ice during the slow addition of sufficient saturated ammonium sulfate solution (pH 7.0 attained by the addition of cone. NH40H, 4 °) to bring the final ammonium sulfate concentration to 0.55 saturation. 16 Thus, to every 100 ml of enzyme solution, 122 ml of cold neutral saturated ammonium sulfate is added. The mixture is centrifuged as before, the precipitate is discarded, and the supernatant fraction is brought to 0.75 saturation. The precipitate is recovered by centrifugation, dissolved in 0.050M phosphate-0.010M 2-mercaptoethanol, pH 7 (about onetenth the orginal volume), and dialyzed for 4 hours at 4 ° against 100 volumes of the buffered mercaptoethanol. Alumina Cy Gel Fr,actionation. The pH of the dialyzed solution is adjusted to 5.5 with 0.05 M sodium acetate, pH 4.0. Trials must be performed with alumina C,/ gel (20 mg/ml) to determine the minimum volume of gel required to adsorb 90% of the enzyme during an exposure of 20 minutes at 4 ° . Following treatment of the enzyme batch with the calculated amount of gel, the gel is recovered by centrifugation and washed with 0.005 M phosphate-0.002 M 2-mercaptoethanol, pH 5.5. The enzyme is eluted with 0.20 M phosphate-0.020 M 2-mercaptoethanol, pH 7.0 by stirring the gel for 20 minutes at 4 ° , and the gel is removed by centrifugation. The purification is usually 80-fold, recovery 20%. Most preparations are stable for several weeks at 4 ° if not dialyzed. Properties

Specificity.Uridine alone among the pyrimidine ribonucleosides is a substrate; 15 cytidine is not phosphorolyzed, 15 although arabinosyl uracil ~oThe volume of cold saturated a m m o n i u m sulfate (V) to be added to the enzyme solution (volume ~ E) is given approximately by the equation E (D -- S) -- 100 V - DV, wherein D is the desired final percentage saturation and S is the percentage of a m m o n i u m sulfate saturation of the solution of enzyme at the time the addition is made.

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is probably converted at a rate about 1% of the rate observed with uridine, when the arabinosyl uracil is present at 15 times the uridine concentration. TM Thymidine is not phosphorolyzed; and in the direction of synthesis, cytidine or orotidine are not synthesized from cytosine or orotic acid and ribose 1-phosphate, is nor is ribosyl thymine formed from thymine and ribose 1-phosphate. ~ Other pyrimidine bases and ribosides have not been tested. The enzyme thus appears to be quite specific for both the pentose phosphate and the pyrimidine base, at least in comparison with thymidine phosphorylase and uridine (thymidine) phosphorylase of ascites cells. 1 Preparation of Nucleosides and Deoxynucleosides Reaction mixtures containing pyrimidine base and pentose phosphate in 0.005 M Tris plus 0.005 M'2-mercapteethanol, pH 7.5, eventually reach equilibrium when pentoside formation is about 60%. Since pyrimidine pentosides are acid stable, protein may be precipitated with acid, the acidic supernatant fluid treated with Dowex 1 (chloride form) to remove phosphates, and the product of the reaction separated from the base and from any free pentose by chromatography on paper. Solvents of choice for ribosides are water-saturated butanol, or butanol saturated with 4% boric acid. 17 For deoxyribosides, the most difficult separation is between thymine and thymidine, for which ethyl acetate (freshly distilled), formic acid, water (60:35:5, upper phase) is useful? a Preparation of Pentose Phosphates Phosphorolysis of pentosides, at pH 6.0 with thymidine phosphorylase and at pH 7.0 (for maximum enzyme stability) for uridine phosphorylase, proceeds until 30% of the substrate has been converted unless excessive amounts of inorganic phosphate are used; for example, a 10-fold increase in phosphate relative to pentoside concentration yields less than a 3-fold increase in the amount of phosphorolysis. However, inorganic phosphate may be precipitated as MgNH4PO4 and the pentose phosphate separated from the rest of the components by eleetrophoresis, by column or paper chromatography, TM or by precipitation. ~, 2~

1~R. Y. Thomson, in "Chromatographic and Electrophoretic Techniques" (I. Smith, ed.), p. 237. Wiley (Interscience), New York, 1960. is K. Fink, R. E. Cline, R. B. Henderson, and R. M. Fink, J. Biol. Chem. 221, 425 (1956). 19A. A. Benson, Vol. III [15]. P. E. Plesner and H. Klenow, Vol. III [25]. 21M. Friedkin and H. M. Kalckar, "Col. III [26].