Reversible ATP-dependent Transition between Two Forms of Human ...

Vol. 268, No. 21, Issue of July 25, pp. 15621-15625.1993 Printed in U . S . A .

THEJOURNALOF BIOLOGICAL CHEMISTRY

0 1993 by The American Society for Biochemistry and Molecular Biology, h

.

Reversible ATP-dependent Transition between TwoForms of Human Cytosolic Thymidine Kinase with DifferentEnzymatic Properties* (Received for publication, October 14, 1992, and in revised form, February 23, 1993)

Birgitte Munch-Petersen$$,Gerda Tyrstedll, and Lisbeth Cloosll From the $Department of Biology and Chemistry, Roskilde Uniuersity, DK 4000 Roskilde, Denmark and the lDepartment of Medical Biochemistry & Genetics, Biochemistry Laboratory C, Panum Institute, Copenhagen Uniuersity, D K 2200 N Copenhagen, Denmark

1976; Munch-Petersen and Tyrsted, 1977; Piper et al., 1980; Sherley andKelly, 1988a). Several investigations haveshown that the changes in TK1 mRNA during cell the cycle are too limited to accountfor the pronounced fluctuations in enzyme activity (Coppock and Pardee,1987; Sherley and Kelly, 1988b; Ito and Conrad, 1990). This indicates that translational and posttranslational modifications are predominantregulating in thymidine kinase activity in cycling cells. Recently, it has been shown that amino acid residues near the C-terminal end are responsible for degradation of thymidine kinase protein in the Gz and M phase, and that mutations in this part of the gene allow expression in Go cells (Kauffman and Kelly, 1991; Kauffman et al., 1991). During the years, there havebeen many reports on the properties of human TK1, with diverging results and observations. Nativemolecular weights between45,000 and 200,000 have been reported for the enzyme from leukemic cells (Lee and Cheng,1976a; Sherley andKelly, 1988a; Munch-Petersen, 1990), human placenta (Ellims et al., 1982; Gan et al., 1983; Tamiya et al., 1989),and lymphocytes (Munch-Petersen, 1984; Munch-Petersen et al., 1991). There have been indicationsthatATP inducespolymerization of the enzyme (Munch-Petersen, 1984; Tamiya et al., 1989; Munch-Petersen et al., 1991), and cooperative and non-cooperative substrate and inhibition kinetics have been reported (Lee and Cheng, 197613; Gan et al., 1983; Munch-Petersen, 1984). The divergent The cytosolic thymidine kinase, TK1‘ (ATP:thymidine5 ’ - results may be due to differentdegrees of purification or the phosphotransferase, EC 2.7.1.21) is a cell cycle-regulated en- presence of substrates. Thus, addition of ATP or thymidine zyme in the nucleoside salvage pathway rescuing thymidine often was used for stabilization of the otherwise very labile from extra- and intracellular catabolic processes. Despite a enzyme (LeeandCheng, 1976a, 1976b; Gan et al., 1983; considerable number of investigations, the exact role of the Munch-Petersen, 1984; Munch-Petersen et al., 1991). Reenzyme has not yet been clarified. Aclose correlation between cently, T K 1 waspurified to homogeneityfrom HeLa cells the thymidine kinase activity and the proliferative state of (Sherleyand Kelly,1988a) and from human lymphocytes the cell has been established, and the fluctuation of thymidine (Munch-Petersen et al., 1991), and in both reports, the native kinase activity during thecell cycle is more pronounced than enzyme in the presence of ATP was a tetramer of a 24-kDa of other enzymes associated with the DNA synthesis (Kit, polypeptide. T o clarify the previously reported divergences, we decided to study the kinetics on the pure thymidine kinase *This work was supported by the Danish Cancer Society, the after removal of substrates anddiscovered that the enzymatic Danish Research Council, NOVO Research Foundation, Otto Johanproperties of thesubstrate-free enzymedifferedmarkedly nes Bruun Foundation,P. Carl Petersens Foundation, and The Foundation for Protection of Animals. The costs of publication of this from previous observations. Furthermore, we found that inarticle were defrayed in part by the payment of page charges. This cubation of the substrate-free enzyme with ATP induced a article must therefore he hereby marked “aduertisement” in accord- slow transition to a more active kinase with more than 20ance with 18 U.S.C. Section 1734 solely to indicate this fact. fold higher substrate affinity.Removal and readditionof ATP 3 T o whom correspondence should be addressed: Dept. of Biology showed that the effect of ATP on the kinetic behavior and and Chemistry ( l ) , Roskilde University, Box 260, DK-4000 Roskilde, the substrate affinitywas completely reversible. Denmark. Tel.: 45-46 75 77 11 (ext. 2418); Fax: 45-46 75 77 21.

Human cytosolic thymidine kinase, subunit molecular mass about 24 kDa, isa tetramer in the presence of ATP but a dimer in the presence of thymidine or without substrates. The pure, substrate-free enzyme showed complex, non-hyperbolic thymidine substrate kinetics withan apparent K , of 15 p ~ Incubation . with ATP at 4 “C induced a time-dependent transition to an enzyme form with hyperbolic kinetics and a 20-fold lower K, value for thymidine (0.7 @M) but the same maximal velocity as for cytosolic thymidine kinase (TKl) without ATP. Removal of the ATP by carboxymethyl chromatography reestablished the non-hyperbolic kineticswith the lowaffinity for thymidine (Km(npp) = 12 p ~ ) and , this enzyme form could be reversed once more by ATPincubation to the high affinity enzyme form. Similar shifts could not be induced by thymidine. The activating effect of ATP depended on the concentration of enzyme protein in a linear manner. These results indicate that ATP is a positive effector of cytosolic thymidine kinase, controlling a kineticallyslow transition between two molecular forms of the enzyme. A hypothetical reaction mechanism is presented to explain the complex kinetic behavior.

‘The abbreviations used are:TK1, cytosolic thymidine kinase; EXPERIMENTAL PROCEDURES CHAPS, 3-[(3-cbolamidopropyl)dimethylammonio]-l-propanesulfonate; TK1-ATP, substrate-free TK1 resulting from removal of thyChemicals-CHAPS was purchased from Boehringer Mannheim, midine by CM-Sepharose chromatography; TKl+ATP, TKl-ATp CM-Sepharose fast flow from Pharmacia, and unlabeled nucleosides stored in 2.5 mM ATP; TKl-ATP/Z, TKl+ATP after removal of and nucleotidesfrom Servaand Sigma. All otherreagents were ATP by CM-Sepharose chromatography; TKl+ATP/2, TKl-ATP/Z commercial preparations of the highest purity available. [ n ~ e t h y l - ~ H ] stored in 2.5 mM ATP. Thymidine (925 GBq/mmol) was obtained from the Radiochemical

15621

15622

ATP Controlled Shifts between Two Forms

Centre, Amersham. Only batches less than 2 months old were used for kinetic experiments. Buffers-Buffers used were as follows. Buffer A: 50 mM potassium phosphate buffer (pH 7.5) containing 0.25 M sucrose, 5 mM dithiothreitol, 5 mM benzamidine, and 0.5 mM phenylmethanesulfonyl fluoride. Buffer B: 25 mM Tris-HC1 (pH 7.6), 20% glycerol, 5 mM MgC12,50 mM mercaptoethanol. Buffer C: 10 mM Tris-HC1 (pH 7.6), 10% glycerol, 5 mM MgCI,, 5 mM dithiothreitol, 0.5 mM CHAPS, and 200 pM thymidine. Buffer D: 10 mM Tris-HC1 (pH 7), 10% glycerol, 5 mM MgCIz, 5 mM dithiothreitol. Buffer E: 50 mM Tris-HC1 (pH 8), 10% glycerol, 5 mM MgCI2, 5 mM dithiothreitol, 0.5 mM CHAPS, and 0.1 M KC]. Buffer F 50 mM Tris-HCI (pH 7.4), 5 mM MgCI,, 3 mM mercaptoethanol, and 0.1 M KCI. Thymidine Kinase Assay-The thymidine kinase activity was assayed by the DEAE-cellulose 81 paper method as previously described (Munch-Petersen, 1984). Standard assay conditions were: 50 mM Tris-HC1, pH 8.0 (22 "C), 2.5 mM MgCI2, 10 mM dithiothreitol, 3 mg/ ml bovine serum albumin,2.5 mM ATP, 10 p~ radiolabeled thymidine (67 GBq/mmol), and 0.1 ng of TK1/50 pl of reaction mixture, unless otherwise specified. Samples of 10 p l were spotted on the DEAE papers 0.5, 5, 10, and 15 min after addition of enzyme extract to prewarmed (37 "C) assay mixture.The filters were eluted and counted as described (Munch-Petersen etal., 1991). The progress curves were linear for at least 60 min when less than 10%of the initial substrate concentration was converted to product. One unit of enzyme activity is defined as the amount of enzyme that can phosphorylate 1 pmol of nucleoside per min at 37 "C under standard assay conditions. Kinetics-The substrate kinetics were analyzed by double-reciprocal plots ( l / u uersm l/s), Hofstee plots ( u uersm u / s ) , and Wilkinson plots (s/uuersus s). u is the initialvelocity, and s is the substrate concentration. The degree of apparent cooperativity was analyzed by Hill plots of log(u/( VmaX- u ) ) = nlog s - nlog So.s, where n is the Hill coefficient and So.6the substrate concentration athalf-maximal velocity ( VmaJ. VmaXand So.&were calculated using non-linear regression analysis to obtain the best fit between the experimental data and theexpression,

of Thymidine Kinase

1

I

1

I

+++:

/I

+

05

40

50

60 70 80 Fraction number

90

100

FIG.1. The effect ofATP on the apparent native molecular weight of TK1.0.3 ng of pure, substrate-free TK1 (TK1-ATP) was injected on the Superose 12column preequilibrated with buffer F (0). 0.3 ng of TK1-ATP was preincubated for 30 min with 2 mM ATP prior to injection on the Superose 12 column preequilibrated with buffer F containing 2 mM ATP (0).The thymidine kinase activity in the fractions was determined as described under "Experimental Procedures.'' Before each experiment, the column was rinsed with more than 10 column volumes of buffer F. The molecular mass markers (+) are from Left to right: alcohol dehydrogenase, 150 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 29 kDa; and cytochrome c, 12.4 kD. Inset, relation between M , and K A V = (u, - ua)/(uc - ua) for the five marker proteins (0); KAVfor TK1ATP and TKl+ATP (0).ue, UO, and u, are the elution volume, the void volume, and the bed volume, respectively.

theabsence of ATP, the peak of activityeluted with an apparent molecular weight of 56,000. The presence of 2 mM ATP during chromatography increased the apparent molecular weight from 56,000 to 120,000. Since the subunitmolecular mass of TK1 is 24-26 kDa, the native enzyme can exist AsZ + Bs as a dimer or tetramer. The elution profiles are notcompletely u= (Eq. 1) s2 -k cs f D homogenous, and a shoulder at the front of the TK1peak, as of ATP, anda tail seenwhen where s is the concentration of the varied substrate and A, B, C , and seen when eluted in the absence eluted inpresence of ATP may reflect an equilibrium between D are constants. A is used as an estimateof Vmax. Enzyme Preparation-TK1 was purified to homogeneity as de- a dimer and a tetramer form of the enzyme. scribed previously from about 15 X lo9 phytohemagglutinin-stimuSubstrate Kinetics-For characterizingand comparing lated lymphocytes, isolated from 500-mi portions of blood from 24 specificity of T K 1 toward different substrates and inhibitors, healthy donors (Munch-Petersen et aL, 1991). Briefly, the supernaand for clarifying the molecular mechanism, it is necessary to tant from streptomycin-precipitatedcrude homogenized extract (buffer A) was precipitatedwithammoniumsulfate,desalted on perform the experiments on substrate-freeenzyme. Previous Sephadex G-25 (buffer B), separated from TK2 by DEAE chromaattemptsto remove thymidine frompurified T K 1 by gel tography (buffer B), and chromatographed on 3"dTMP-Sepharose filtration resulted in great loss of enzyme activity probably (buffer C). due to dilution of the enzyme protein. In the present work, CM-Sephnrose Chromatography-The purified TK1 was concenwe used CM-Sepharose chromatographyby which thymidine trated and thymidine removed by application of the peak fractions was removed and the enzyme concentrated in a single step, from the affinity chromatography, on a 10 X 20-mm column packed with CM-Sepharose and preequilibrated with buffer D. Thymidine improvingthe yield considerably. Furthermore,immediate was completely removed by washing the column with about20 column addition of ATP to a final concentration of2.5 mM to an volumes of buffer D, as controlled by including [methyL3H]thymidine aliquot of the eluted TK1 increased the yield more than 2in the applied sample in some experiments. The thymidine kinase T K 1 storedinATP is designated activity was eluted with buffer E, and more than 75% of the activity fold. Hereafter,the was eluted in 1 ml. This substrate-free enzyme form is referred to as TKI+ATP, and the substrate-free TK1is designated TK1TK1-ATP. Immediately after elution of the activity, ATP was added ATP. to an aliquot of the eluted activity to a final concentration of 2.5 mM. For further studying the effect of ATP on the enzyme, we The thymidinekinaseactivitystored in ATP is referred toas incubated substrate-free TK1 at a concentration of 200 ng/ TKl+ATP. The yield of activity was 40-60%, if more than 1 pg of enzyme was applied, but decreased when the applied amount was ml, with 2.5 mM ATP with or without6 p~ thymidine at 4 "c decreased. In analyticalexperiments, bovine serumalbumin was for various time intervals, andassayed the enzyme activity at added to stabilize the enzyme. Removal of ATP from TKl+ATP was 37 "C.A typical result of such an experimentis shown in Fig. performed by the same procedure. 2 and demonstratesa more than 3-fold increase in thymidine Molecular Weight Determination-The apparent molecular weight kinaseactivityafter 2h of incubation. Furthermore,the of native TK1 was determined as previously described on a Superose activation. 12 column (10 x 300 mm) connected t o a fast protein liquid chro- presence of thymidine seems to increase the ofrate no activation was obtained when incubatOn the other hand, matography system (Pharmacia) (Munch-Petersen etal., 1991). ing the enzyme with thymidine alone or without substrates. RESULTS The activationwas strongly dependent ina linear manner on the concentration of enzyme protein (Fig. 2, inset), and no Molecular Weight Properties-The effect of ATP on the native molecular weight of T K 1 is demonstrated inFig. 1. In activation was obtained below 10 ng/ml. This implies that no

15623

ATP Controlled Shifts between Two Forms of Thymidine Kinase 1 I

12,

-

50

0

5

0

100

min of incubation with ATP

FIG. 2. The effect of ATP on the activity of TK1. 20 ng of pure, substrate-free TK1 (TK1-ATP)was incubated at 4 "C in 0.1 ml of buffer E containing 2.5 mM ATP (0)or 2.5 mM ATP plus 6 PM thymidine (0).At the indicated times, aliquots of the incubation mixture were diluted 50-fold in buffer E and assayed at 37 "C for thymidine kinase activity with 1 p~ thymidine at standard assay conditions. Inset, TK1-ATP were incubated 1h at the indicated enzyme concentrations at 4 "C in buffer E containing 2.5 mM ATP. The enzyme activity was assayed at 37 "C by addition of 0.1 ng of the incubated enzyme to 50 p1 of standard reaction mixture.

v/[dThd]

10

(unlts/mg/vM)

FIG. 4. The effect of ATP on the thymidine substrate kinetics of TK1. Hofstee plots of the initial velocities of TKl-tATP (O), TK1-ATP (O),TK1-ATP incubated for 60 min at 4 "C with 2.5 mM at various thymidine ( d T M ) ATP (A), or with 100 pM thymidine (a) concentrations are shown. Hill plots of the data are shown in the inset.

the incubated enzyme measured at low thymidine concentrations were increased about %fold, whereas at saturating thymidine concentrations the initial velocities of TK1-ATP and the incubated TK1 were almost equal. Further storage of the I I. I incubated enzyme in the incubation mixture at -70 "C for 2 weeks resulted in complete reversal to the kinetics obtained 1 I I with TKl+ATP, since the straight line in the Hofstee plot superimposed the line obtained with TKl+ATP. From Hill plots of the data (Fig. 4, inset), So., values of 0.7 and 15 PM were calculated for TKl+ATP and TK1-ATP, respectively, indicating that TKl+ATP has a more than 20-fold higher affinity for thymidine than TK1-ATP. The Hill coefficient for TK1-ATP was 0.74, indicating an apparent negative cooperative reaction mechanism, but increased to 1.0 after 1 h of incubation with ATP. The Hill coefficient for TKl+ATP 00 was 1.17. 2 4 6 a 0 Incubation of TK1-ATP at 100 p~ thymidine for 2 h at ng o f T K I - A T P 4 "C did not change the thymidine substrate kinetics as indiFIG. 3. The relation between rate of dTMP formation and cated by the biphasic Hofstee plot (Fig. 4). The Hill plot of concentration of TK1-ATP at different thymidine concentrations. The initial velocities at the indicated amounts of TK1-ATP these data superimposed the plot obtained with TK1-ATP, per 50 p1 of assay mixture were measured at 0.1 pM (a),1 p~ (m), and So.5was14 WM and the Hill coefficient was0.74. The biphasic kinetic pattern was retained during further storage and 10 p M (A) thymidine. in thymidine. We then examined the effect of removing ATP from activation takes place at thestandard assay concentration of enzyme which is 2 ng/ml. TKl+ATP by a second chromatography on CM-Sepharose. The relation between rate of product formation and amount An aliquot of the resulting, substrate-free TK1, designated of enzyme protein in the reaction mixture was linear with TKl-ATP/2, was made 2.5 mM with ATP and designated TKl+ATP but non-linear with TK1-ATP atthymidine con- TKl+ATP/2. Similar kinetic experiments as those depicted centrations below 10 PM (Fig. 3). The upward concave curves in Fig. 4 were performed with the rechromatographed enzyme may be explained by a protein concentration-dependent for- forms, and theresults areshown in Fig. 5 . The Hofstee curves mation of a more active enzyme complex. and the Hill plots clearly indicate that the effect of ATP on Fig. 4 demonstrates that storage orincubation of TK1 with TK1 is reversible, since removal of ATP reestablished the ATP has a strikingeffect on thereaction mechanism between blphasic pattern with the low thymidine affinity, and asecond TK1 and thymidine, as analyzed by measuring the initial incubation with ATP reestablished the rectangular hyperbolic velocity at various assay concentrations of thymidine at a pattern with the high thymidine affinity. The So.svalues and fixed saturating assay concentration of ATP. As can be seen Hill coefficients were 1 2 WM and 0.73 for TKl-ATP/Z, 1.4 WM from the Hofstee plots, TK1-ATP exhibited a biphasic kinetic and 0.96 for TKl-ATPIB incubated with ATP, and 1 p~ and pattern, whereas the straight line obtained with TKl+ATP 1.0 for TKl+ATP/Z, respectively. indicates a rectangular hyperbolic reaction mechanism. At The biphasic kinetics of TK1-ATP was retained at various thymidine concentrations below 10 p ~ the , initial velocity assay concentrations of ATP between 0.025 and 5 mM, as with TKl+ATP was more than 3-fold higher than that with indicated by the parallel lines in the Hill plots (Fig. 6) and TK1-ATP, whereas at saturating thymidine concentrations Hill coefficients between 0.75 and 0.8 (Fig. 3). The So, values the velocities are nearly the same for the two enzyme forms. increased from 14 to 130 PM thymidine, when the assay Incubation with ATP for 1 h at 4 "C reversed the biphasic concentration of ATP decreased from 5 to 0.025 mM. kinetics of TK1-ATP to rectangular hyperbolic kinetics as In a similar series of experiments with TKl+ATP (not indicated by the linear Hofstee plot. The initial velocities of shown), the SO.Svalues at ATP concentrations between 5 and

1

E

v 1,

15624

ATP Controlled Shifts between Two Forms of Thymidine Kinase1 10

-

E

\

5 -

05

0

E C

on

FIG. 5. Effects of removal of ATP, and reincubation with ATP on the thymidine substrate kinetics. Hofstee plots of the initial velocities of TKl+ATP/2 (O), TK1-ATPIP (O), and TK1ATP/2 incubated for 60 min at 4 "C with 2.5 mM ATP (A) at various thymidine ( d T h d ) concentrations are shown. Hill plots of the data are shown in the inset. The complete removal of ATP from TKl+ATP by carboxymethyl chromatography was controlled by omitting ATP from the assay mixture.

T >

t-

"-c

-1

3

v

\

>

m

-0

"t log [dThd]

(uM)

FIG. 6. Effects of various assay concentrations of ATP on the thymidine substrate kinetics. The initial velocity of TK1ATP was measured at varying thymidine ( d T h d ) concentrations at fixed assay concentrations of ATP. The data are plotted according to 0.1 mM ATP; A, 0.05 mM ATP; the Hill equation. 0, 5 mM ATP; 0, and A, 0.025 mM ATP.

0.025 mM were almost constant, in the range of 0.5-0.7 p M thymidine. Double-reciprocal plots of the data from these experiments gave an intersecting pattern of straight lines with TKl+ATP, indicating a compulsory order steady-state reaction mechanism, with formation of a ternary complex between the enzyme and the two substrates, as previously observed with a less purified preparation of T K 1 (Lee and Cheng, 1976b). TK1-ATP may also follow a similar but more complex reaction mechanism sincedownward curved doublereciprocal plots (not shown) of the data in Fig. 6 gave an intersecting pattern at high (>20 PM) and low (