[45] Thymidine kinase from Escherichia coli

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polypeptide regions, one for the active site of dCyd kinase (and the effector site for dAdo kinase) and the other for dAdo kinase, could account for these observations.

Characteristics of dGuo/dAdo Kinase The properties of this enzyme preparation are not yet determined, although preliminary studies suggest that they parallel the characteristics of the dCyd/dAdo kinase enzyme.

[45] T h y m i d i n e K i n a s e f r o m E s c h e r i c h i a coli

By MING S. CHEN and WILLIAM H. PRUSOFF Deoxythymidine + NTP--* dTMP + NDP

Thymidine kinase (EC 2.7,1.75) from Escherichia coli is an unusual allosteric enzyme whose most striking characteristic is its regulation not only by the end-product dTTP, but also by a number of nucleoside diand triphosphates.1 Dimerization of the enzyme molecule accounts for the regulatory properties, and this "dimer" molecule has either an active or an inactive conformation, depending on the specific nature of the effector nucleotide with which it interacts.2

Assay M e t h o d

Principle. Enzymic activity has been assayed by conversion of labeled deoxythymidine to deoxythymidine monophosphate, with separation of the substrate and product by either high-voltage paper electrophorcsis,3 column chromatography, 4 thin-layer chromatography, 5 paper chromatography, ~ or disc DEAE-cellulose. r 1 R. Okazaki and A. Kornberg, J. Biol. Chem. 239, 275 (1964). N. Iwatsuki and R. Okazaki, J. Mol. Biol. 29, 139 (1967). 3 R. Okazaki and A. Kornberg, J. Biol. Chem. 239, 269 (1964). 4 p. S. Fitt, P. I. Peterkin, and V. L. Grey, J. Chromatogr. 124, 137 (1976). 5 D. L. Greenman, R. C. Huang, M. Smith, and L. M. Furrow, Anal. Biochem. 31, 348 (1969). 8 W. J. Reeves, Jr., A. S. Seid, and D. M. Greenberg, Anal. Bioehem. 30, 474 (1969). 7 M. B. Furlong, Anal. Biochem. 5, 515 (1963). METHODS IN ENZYMOLOGY, VOL. LI

Copyright© 1978by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181951-5

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Reagents Tris'HC1, buffer 0.2 M, pH 7.8 ATP, sodium salt, 50 mM MgClz, 50 mM Bovine serum albumin (BSA) [2-14C]Thymidine, 4.8 mM, sp act = 0.5-1.5 Ci/mole The stock assay solution consists of 0.7 ml Tris buffer, 0.4 ml of ATP, 0.4 ml MgClz, 0.6 mg BSA, and 0.5 ml [2-14C]thymidine. This mixture can be stored in the cold for up to 1 week. For assaying crude cell homogenates, 20 mM NaF is included to inhibit phosphatase activity.

Procedure. The reaction mixture contained in 0.1 ml of final volume is: 75 ~1 of the above stock assay solution plus 25 tzl of enzyme solution. The conversion of deoxythymidine to deoxythymidine monophosphate was measured by one of the following two procedures: (1) By adsorption onto DEAE discs as described by Furlong. 7 Portions of the reaction mixtures (approximately 20-30 txl) are spotted onto DEAE discs (2 cm in diameter) which are then dropped into a beaker containing about 10 ml of 95% alcohol per disc. The alcohol is decanted, and the discs are resuspended in the same volume of alcohol for 10 min. This washing procedure in 95% alcohol is repeated two times. The discs are then placed on a paper towel, dried in air, and counted in a liquid scintillation counter with toluene-POPOP. (2) By spotting portions of the reaction mixture (approximately 2-5 /~1) onto a cellulose TLC sheet with development in 0.5 M LiCI to a distance of 15 cm. The spot that corresponds to a deoxythymidine monophosphate marker is cut out and counted as in (1). The assay of deoxythymidine monophosphate by TLC has the advantage of detecting potential contamination of the enzyme during purification with deoxythymidine monophosphate kinase, and if also present nucleoside diphosphokinase, since deoxythymidine-diphosphate and -triphosphate have different R e values in the above TLC system. Another advantage of TLC is when [~/-3ZP]ATP is used to study the potential of an agent to be an alternate substrate. The formed monophosphate of a substrate nucleoside analog in this system is readily separated from the phosphate donor (e.g., [y-32p]ATP, R e = 0.06; nucleoside monophosphate, R e = 0.47). Definition of Units of Activity and Specific Activity. One unit of thymidine kinase activity is defined as the amount of enzyme catalyzing the formation of one /zmole of deoxythymidine monophosphate per minute at 37 °. Specific activity is expressed as the number of units of

356

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thymidine kinase activity per milligram of protein. Proteins were determined according to Lowry et al.S Purification P r o c e d u r e The purification procedure has been described by Voytek et al.9 The enzyme solution is maintained at 0 - 4 ° unless otherwise indicated. Step 1. Preparation o f Crude Extract. Frozen E. coli B cells which had been grown in Kornberg's medium and harvested in late log phase were purchased from General Biochemicals and stored at - 7 0 °. The extraction procedure is a modification of the method reported by Okazaki and Kornberg. ~ Cells (500 g) were thawed for 12 hr at 0 ° and then placed in a commercial Waring Blendor that contained 300--400 ml of chilled Tris-HCl buffer (20 mM, pH 7.8) plus EDTA (2 raM). The blender was started at a low speed, and 500 g of glass beads (Superbrite No. 100, Minnesota Mining and Manufacturing Company, Inc.) which had been previously chilled to - 7 0 ° were added slowly. The speed of the blender was increased (blender speed low, rheostat setting 75 V) and maintained for 25 min. During the extraction the temperature remained below 0 °. The suspension was then centrifuged for 30 min at 13,000 g. The supernatant fluid was decanted and subjected to a second centrifugation at 100,000 g for 3 hr. The supernatant liquid obtained was clear and yellow and is designated as fraction 1 in the table. PURIFICATION DATA FOR Escherichia coli THYMIDINE KINASEa

Fraction and step 1. 2. 3. 4. 5. 6.

Extract Streptomycin Ammonium sulfate Sephadex First gel electrophoresis Second gel electrophoresis

Volume (ml)

Units

Protein (mg/ml)

440 415 16 10 16 6

9.27 8.27 2.63 0.77 0.23 0.11

15.8 16.6 34.0 4.4 0.2 0.025

Specific activity (units/mg) Purification × l0 s factor 1.3 1.0 5.0 17.0 78.0 720.0

1.0 0.75 4 12 59 531

a Reproduced from Voytek et a l ? (Table I). The units are expressed as the conversion of 1 /~mole of deoxythymidine to deoxythymidine monophosphate in 1 min at 37 ° rather than in 1 hr as presented in the original table. ~1 s O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 9 p. Voytek, P. K. Chang, and W. H. Prusoff, J. Biol. Chem. 246, 1432 (1971).

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357

Step 2. Streptomycin Sulfate Fractionation. To every 100 ml of fraction 1, 2.63 ml of a 38% streptomycin sulfate solution (Grade B, Calbiochem) were added drop by drop over a 30-min period with stirring. The solution was stirred for an additional 30 min, frozen, stored at - 2 0 ° overnight, thawed, and then centrifuged at 13,000 g for 30 min. The supernatant fluid (fraction 2) was clear yellow. Step 3. Ammonium Sulfate Fractionation. Solid ammonium sulfate was slowly added to fraction 2 to a final saturation of 40%. After additional stirring for 30 rain, the mixture was centrifuged at 13,000 g, and the precipitate was resuspended in 16 ml of 50 mM Tris'HCl buffer (pH 7.8) containing EDTA (0.5 mM) and 2-mercaptoethanol (9 mM). The mixture was dialyzed overnight against the same buffer (fraction 3). Step 4. Sephadex G-150 Chromatography. Fraction 3 was added to a Sephadex G-150 column (2.5 × 100 cm) which had been previously washed with 2 liters of the dialysis buffer. The proteins were eluted from the column by the upward flow technique. The major fractions containing 75% of deoxythymidine kinase activity were pooled and concentrated 8-fold in an Amicon ultrafiltration cell model 202 with a UM-20E filter (fraction 4). Step 5. Preparative Polyacrylamide Disc Gel Electrophoresis I. A Buchler preparative polyacrylamide gel electrophoresis apparatus was used. Sucrose and Bromphenol blue were added to fraction 4 making the final concentrations 3 and 0.001%, respectively. The protein solution was then layered on top of a 4-cm 7.5% gel which was prepared with the use of the Tris-glycine buffer system at pH 8.9 according to Davis. TM MgCI2 (1 mM) was also included in the gels. The upper buffer (pH 8.9) consisted of Tris (53 raM), glycine (53 mM), cysteine (1.2 mM), MgCI2 (1 mM), and 2-mercaptoethanol (20 raM). The composition of the lower buffer was Tris.HCl (100 mM) at pH 8.0 and MgCI~ (1 mM). The elution buffer contained Tris'HCl (100 mM) at pH 8.0, MgC12 (1 mM), cysteine (1.2 raM), and 2-mercaptoethanol (20 mM). The membrane-holder buffer was Tris.HCl (400 raM) at pH 8.1 and MgCI~ (1 mM). A constant current of 60 mA was applied across the gel for approximately 18 hr by which time the deoxythymidine kinase had passed through the gel. The flow rate of the elution buffer was 1 ml per minute. The coolant temperature was maintained at 1°. Eluted fractions containing deoxythymidine kinase activity were pooled and concentrated approximately 8-fold by ultrafiltration (fraction 5). Step 6. Preparative Polyacrylamide Disc Gel Electrophoresis H. The 10 B. J. Davis, Ann. N. Y. Acad. Sci. 121,404 (1964).

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second preparative electrophoresis was performed in a manner identical to the first but modified by use of a 5% polyacrylamide gel at a height of 5 cm. Fractions that contained the enzymic activity were pooled and concentrated as described above (fraction 6). Fraction 6 (25--40 ttg of protein per milliliter) was divided into 0.3-ml aliquots and stored at - 7 0 °. No loss in activity occurred within a 1-month storage period, and there was no change in the response to the nucleoside effectors. The results of a typical preparation are summarized in the table, 9"11 and the enzyme obtained was found to be electrophoretically homogeneous. Properties

Substrate Specificity. The enzyme can use the following as a phosphate acceptor: deoxythymidine, deoxyuridine, 5-halogenated analogs (5-fluoro-, 5-chloro-, 5-bromo-, and 5-iodo-deoxyuridineS), and 5mercaptodeoxyuridine. 12 Like other nucleoside kinases, the enzyme has a broad specificity with regard to the phosphate donor; most nucleotide triphosphates with the exception of dTTP have been found able to donate phosphate in the reaction. The ability to serve as a phosphate donor follows the following order; ATP, dGTP, dATP, dCTP, GTP, and ITP. Other nucleoside triphosphates and all nucleoside diphosphates are inert as phosphate donors. The metal ion requirement can be fulfilled by either Mg 2+ or Mn 2+. p H Optimum. Activity as a function of pH is maximal at about pH 7.5. Stability. The enzyme appears to be stable for several months if kept at - 7 0 ° in the presence of l0 mM fl-mercaptoethanol and added BSA (0.5 mg/mi). Molecular and Allosteric Regulatory Properties. E. coli thymidine kinase is an allosteric enzyme. 1 Its activity is not only regulated by the end-product dTTP, but also by a number of nucleoside di- and triphosphate. The end-product dTTP is an inhibitor, while the following nucleotides are activators: dCTP > dATP > dGTP > dCDP > hydroxymethyl-dCTP > hydroxymethyl-dCDP > GTP > dADP > dGDP > GDP. 1 The halogenated analogs 5-iodo-dCTP and 5-bromo-dCTP are more potent allosteric activators than the naturally occurring dCTP. 9 These nucleoside di- and triphosphates, whether inhibitory or activating, ~1p. Voytek, P. K. Chang, and W. H. Prusoff,J. Biol. Chem. 247, 367 (1972). lz T. I. Kalmanand T. J. Bardos, Mol. Pharmacol. 6, 621 (1970).

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exert their regulatory control of the enzyme activity by an aUosteric phenomenon in which the initial event appears to be the dimerization of the enzyme, z Those allosteric regulators that are activators increase the sedimentation coefficient from 3.4-3.5 S to 5.3-5.6 S, whereas those that inhibit increase the sedimentation coefficient to 5.9-6.0. 9 Whereas 5-iodo-dUTP is a very potent activator at pH 7.8, replacement of the oxygen in the 5'-position of IdUTP with an imino (-NH-) moiety produced not only an inhibitory effector but also one that is 60-fold more potent than dTTP as allosteric regulator.13 In general, the effect of activating effectors is to decrease the Km for substrate and increase the Vm of the reaction, while the inhibiting effectors increase the Km and decrease the Vm. The molecular weight of the "monomer" and " d i m e r " is 42,000 and 90,000, respectively. 2 In addition to its role as a phosphate donor, ATP at high concentrations behaves as an activator, and hence its presence normalizes the plot of Michaelis-Menten kinetics of thymidine phosphorylation from sigmoidal to hyperbolic in shape. 1 The monomer form of the enzyme is more sensitive to temperature 14 and ultraviolet (UV) irradiation 15 than when in the dimer form (in the presence of a naturally occurring regulatory nucleotide). When the dimer is induced by 5-iodo-dUTP or 5-iodo-dCTP the enzyme is more sensitive to UV irradiation than when in the monomer form (Chen and Prusoff, unpublished result). The enzyme is protected against UV inactivation by the normal substrate deoxythymidine, whereas the halogenated analog of thymidine, 5-iododeoxyuridine (an alternate substrate), enhances the rate of UV inactivation, ua5 The formation of a free radical in the 5position of the pyrimidine moiety during radiation-induced dehalogenation of 5-iodo analogs of either the alternate substrate or regulatory nucleotides accounts for such enhanced UV sensitization of the enzyme. [14C]-Labeled IdUrd or IdUTP when irradiated in the presence of the enzyme forms a covalent linkage with the protein which is thus inactivated. K i n e t i c C o n s t a n t s . aa~ The K m for deoxythymidine is 1.7 × 10-SM and for 5-iodo-2'-deoxyuridine 1.2 × 10-5 M. The Ki for 3-N-methyl-5iodo-2'-deoxyuridine is 1.7 x 10-a M. Since ATP also regulates the enzyme activity as an allosteric activator at high concentration, the above kinetic constants will depend on the ATP concentration? Km values of the activating regulators are 5 x 10-6 M (5-iodo-dCTP, pH 5.5), 22 x 10-nM (5-bromo-dCTP, pH 5.5), 29 x 10-nM (dCTP, pH 13 M. S. Chen, D. C. Ward, and W. H. Prusoff, J. Biol. Chem. 251, 4839 (1976). 14 N. Iwatsuki and R. Okazaki, J. Mol. Biol. 29, 155 (1967). 15 R. Cysyk and W. H. Prusoff, J, Biol. Chem. 247, 2522 (1972).

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5.5), 4 × 10-SM (5-iodo-dCTP, pH 7.4), 14 x 10-6M (5-bromo-dCTP, pH 7.4), 27 × 10-6M (dCTP, pH 7.4), and 10 x 10-6 M (5-iodo-dUTP, pH 7.4). The Km value for the inhibitory effector dTTP is 16 × 10-5 M, and for 5'-triphosphate of 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUTP) it is 0.67/zM.

[46] D e o x y t h y m i d i n e

K i n a s e in R e g e n e r a t i n g

Rat Liver

By EDWARD BRESNICK Mg2 ÷

Deoxythymidine + ATP

~ d-TMP + ATP

Introduction Deoxythymidine kinase (TdR kinase; ATP-thymidine 5'-phosphotransferase, EC 2.7.1.21) catalyzes the phosphorylation of deoxythymidine and a number of its analogs to form the corresponding deoxyribonucleotides, e.g., d-TMP, at the expense of ATP; this reaction requires a divalent cation, Mg ~+. The enzyme is important in that it is responsible for recycling endogenous deoxythymidine via the pyrimidine salvage pathway and for utilization of exogenous deoxythymidine within tissues. In addition, TdR kinase fulfills an important role in the activation of a number of pyrimidine analogs for chemotherapeutic efficacy, particularly the halogenated pyrimidine deoxyribonucleosides.1 TdR kinase activity within mammalian cells is closely correlated with their proliferation capacity. In this respect, enzyme activity is markedly elevated in regenerating liver, z neoplastic tissues, 3-8 in viraUy infected 1 E. Bresnick, in "The Molecular Biology of Cancer" (H. Busch, ed.), p. 277. Academic Press, New York, 1973. 2 E. Bresnick, Methods CancerRes. 6, 347 (1971). a T. W. Sneider, V. R. Potter, and H. P. Morris, CancerRes. 29, 40 (1969). 4 E. Bresnick and U. B. Thompson, J. Biol. Chem. 240, 3967 (1965). 5 j. Bukovsky and J. S. Roth, Cancer Res. 25, 358 (1965). 6 T. Hashimoto, T. Arima, H. Okuda, and S. Fujii, Cancer Res. 32, 67 (1972). r E. Bresnick, U. B. Thompson, H. P. Morris, and A. G. Liehelt, Biochem. Biophys. Res. Commun. 16, 278 (1964). a E. Bresnick and R. J. Karjala, Cancer Res. 24, 841 (1964). METHODS

IN ENZYMOLOGY,

VOL. LI

Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5