Reversible tetramerization of human TK1 to the high catalytic efficient form is induced by pyrophosphate, in addition to tripolyphosphates, or high enzyme concentration Birgitte Munch-Petersen Department of Science, Systems and Models, Roskilde University, Denmark For decades, it has been the general belief that the building blocks of DNA, the deoxyribonucleoside triphosphates (dNTPs), play a central role in maintai- ning correct DNA synthesis. Recent investigations of DNA synthetic processes in yeast and human cells have indicated that initiation and progress of DNA replication are closely associated with the cellular dNTP concentration [1–3]. The level of the dNTPs is strictly controlled and fluctuates during the cell cycle, in close correlation with the rate of DNA synthesis, with low dNTP levels in G 1 cells increasing during S phase, generally with dTTP being the most abundant and dGTP the least [4–6]. In quiescent cells, dNTP levels are several-fold lower [7], and in non-proliferating human lymphocytes, which are G 0 cells, the dTTP pool is many times smaller than the other dNTP pools [4]. In most cells and organisms except for a few para- sites, the dNTPs are provided by two main routes, the de novo and the salvage pathways. The central enzyme in the de novo route, ribonucleotide reductase, cata- lyzes reduction of ribonucleotides to the corresponding 2¢-deoxyribonucleotides, after which they are phos- phorylated to the triphosphate level by nucleoside diphosphate kinase. The specificity of ribonucleotide reductase is controlled by the concentration of the end-products dATP, dTTP and dGTP, where dTTP is the key regulator switching the specificity from reduc- tion of pyrimidine ribonucleotides to reduction of purine ribonucleotides [8]. Therefore, the cellular dTTP Keywords ATP; gel filtration; kinetics; tetramerization; thymidine kinase Correspondence B. Munch-Petersen, Department of Science, Systems and Models, Universitetsvej 1, Building 18.1, Roskilde University, DK-4000 Roskilde, Denmark Fax: +45 4674 3011 Tel: +45 4674 2419 E-mail: bmp@ruc.dk Website: http://www.ruc.dk/nsm/ (Received 5 August 2008, revised 5 November 2008, accepted 17 November 2008) doi:10.1111/j.1742-4658.2008.06804.x Thymidine kinase (TK1) is a key enzyme in the salvage pathway of deoxy- ribonucleotide metabolism, catalyzing the first step in the synthesis of dTTP by transfer of a c-phosphate group from a nucleoside triphosphate to the 5¢-hydroxyl group of thymidine, forming dTMP. Human TK1 is cytosolic and its activity is absent in resting cells, appears in late G 1 , increases in S phase coinciding with the increase in DNA synthesis, and disappears during mitosis. The fluctuation of TK1 through the cell cycle is important in providing a balanced supply of dTTP for DNA replication, and is partly due to regulation of TK1 expression at the transcriptional level. However, TK1 is a regulatory enzyme that can interchange between its dimeric and tetrameric forms, which have low and high catalytic effi- ciencies, respectively, depending on pre-assay incubation with ATP. Here, the part of ATP that is necessary for tetramerization and how the reaction velocity is influenced by the enzyme concentration are determined. The results show that only two or three of the phosphate groups of ATP are necessary for tetramerization, and that kinetics and tetramerization are closely related. Furthermore, the enzyme concentration was found to have a pivotal effect on catalytic efficiency. Abbreviations dNTP, deoxyribonucleoside triphosphate; dThd, thymidine; hTK1, human cytosolic thymidine kinase 1; NaP, sodium orthophosphate; NaPP, sodium dipolyphosphate; NaPPP, sodium tripolyphosphate; rhTK1, recombinant human TK1; TmTK, TK from Thermotoga maritima. FEBS Journal 276 (2009) 571–580 ª 2008 The Author Journal compilation ª 2008 FEBS 571 level is critical for maintaining a proper balance between the dNTPs. In addition to the ribonucleotide reductase-controlled pathway, the dTTP level is con- trolled by thymidine kinases and TMP nucleotidases, forming a substrate cycle [8,9]. A crucial step in dTTP synthesis is phosphorylation of thymidine (dThd) to dTMP. Two thymidine kinases catalyze this step, the cytosolic TK1 and the mitochon- drial TK2 (EC 2.7.1.21 for both TK1 and TK2), encoded by two nuclear genes. TK1 is cell-cycle-specific and is not expressed in quiescent cells, in which only the constitutively expressed TK2 is present. The complex transcriptional and translational regulation of TK1 ensures that the increase in TK1 activity coincides with an increase in the DNA synthesis rate and dNTP pools [10]. TK1 is degraded to undetectable levels during mitosis by means of the anaphase-promoting complex APC ⁄ C-Cdh1, which recognizes a KEN box in the C-terminus [11]. Human TK1 (hTK1) is a regulatory enzyme that can occur in two forms, a dimer with low activity and a tetramer with high activity. The conver- sion between the two forms is reversible and depends on enzyme concentration and the presence of ATP [12]. When hTK1 purified from human lymphocytes was incubated with ATP prior to assay, the kinetics is hyper- bolic, with a K m of approximately 0.5 lm and a V max of 10 lmolÆmin )1 Æmg )1 . Without pre-assay incubation with ATP, the V max is the same but the kinetics is non-hyper- bolic, with an apparent K m of 15–17 lm and a Hill coef- ficient less than one, indicating negative co-operativity. This behavior means that the catalytic efficiency (k cat ⁄ K m ) is approximately 30-fold higher for hTK1 that had been incubated with ATP. This ‘ATP effect’ on the kinetics apparently depends on the enzyme concentra- tion in a linear manner, and no transition to the catalyti- cally highly active form was observed at concentrations of hTK1 below 10 ngÆmL )1 (0.4 nm) [12]. Therefore, transition does not occur at the low assay concentration of TK1 (< 3 ngÆmL )1 ). This also explains why both enzyme forms showed linear progress curves for product versus time. It is very likely that the ‘ATP effect’ is a fine tuning of the hTK1 activity during the cell cycle. When hTK1 is degraded in G 2 ⁄ M phase, and given that ATP is fairly constant during the cell cycle, the initial low hTK1 concentration in the following G 1 phase implies predominance of the low-activity dimer form. As the hTK1 concentration increases during S phase, more and more enzyme will be in the high-active tetramer form. Recently, phosphorylation of hTK1 at serine 13 has been proposed to be involved in this regulation, preventing ATP-induced transformation to the high- active tetramer [13]. The structure of human TK1 was solved in 2004 [14], and it is closely related to several bacterial TK1 structures but is fundamentally different from the structures of the non-TK1 like kinases deoxycytidine kinase [15], deoxyguanosine kinase and Drosoph- ila melanogaster multi-substrate kinase [16]. This indi- cates a different evolutionary origin of the two classes of deoxyribonucleoside kinases. However, the exact binding of ATP is not clear, as the enzyme is a tetramer with dTTP in the active site for all TK1 structures except the structure for TK1 from Thermo- toga maritima (TmTK) which has the inhibitor TP4A bound to the tetrameric enzyme [17]. The structure of hTK1 with TP4A has also been solved, but here no electron density was seen with adenosine. In TmTK, the adenosine moiety was bound at the a-helix dimer interface, and this form is more open than hTK1. Therefore, at present, it appears that the adenosine group is very loosely bound to hTK1. In the present work, the part of the phosphate donor that is necessary for the dimer–tetramer transi- tion of native hTK1 purified from human lymphocytes was identified. Further, the effect of the concentration of the recombinant enzyme on its oligomerization behaviour was investigated. The results show that the dipolyphosphate group is sufficient for inducing transi- tion to the high-active tetramer, and that kinetics and oligomerization are closely related. In addition, the results show a clear relationship between the enzyme concentration and the catalytically high-active tetra- meric form, and that the tetramer dissociates into dimers very slowly. Results and Discussion Identification of the group inducing tetramerization of human TK1 Human TK1 has 234 amino acids and a subunit size of 25.5 kDa [18]. Several reports have shown by gel filtration that native as well as recombinant hTK1 elutes as a dimer in the absence of ATP (1–5 mm) and as a tetramer in its presence [12,13,19,20]. The recently solved structures of a number of TK1-like enzymes from human, bacteria and vaccinia virus all show tet- rameric forms [14,17,21–23]. As the adenosine moiety does not show electron density in any of the human TK1 structures, it may be that the adenosine moiety is of no significance for inducing the reversible dimer– tetramer transition. Therefore, the present study aimed to identify the part of the nucleotide molecule that triggers tetramerization. Figure 1A–C shows the elution profiles of native TK1 from human lympho- Enzymatic regulation of human TK1 B. Munch-Petersen 572 FEBS Journal 276 (2009) 571–580 ª 2008 The Author Journal compilation ª 2008 FEBS cytes in the presence of the ribonucleoside triphos- phates GTP, CTP and UTP. For comparison, elution profiles with and without ATP are shown in Fig. 1D,E. With all NTPs, hTK1 elutes as tetramers with apparent molecular masses of approximately 100– 120 kDa (see Table 1). Therefore, it can be concluded that the nature of the base is insignificant for the tetra- merization effect. The next goal was to determine the role played by the sugar and phosphate groups. As seen in Fig. 1F, ADP was able to induce the tetramer, whereas, in the presence of AMP, the majority of the enzyme eluted as a dimer with a size of approximately 53 kDa. A minor shoulder is seen at approximately 115 kDa (Fig. 1G) (Table 1). This suggested that the phosphate part of the nucleotide is more important for tetramerization than the sugar and base. Indeed, as seen in Fig. 1H, hTK1 elutes as a tetramer in the presence of sodium tripolyphosphate (NaPPP). In all these elutions, 2 mm MgCl 2 was present in the elution buffers. To determine the effect of sodium dipolyphosphate (NaPP), the gel filtration has to be performed in absence of MgCl 2 , as the combination of MgCl 2 and Chaps causes a heavy precipitate. Fig. 1. Effect of nucleotides and polyphos- phates on oligomerization of native hTK1. Approximately 10 ng native TK1 purified from human lymphocytes in a total volume of 200 lL was injected into a Superdex 200 column (10 · 300 mm) together with 0.1 mg Blue Dextran used as an internal standard for determination of the void volume, V 0 , in the individual experiments. Prior to injection, hTK1 was diluted to 6 lgÆmL )1 and incubated with 3 mM of the indicated nucleotides or polyphosphates at 4 °C for 2 h, and stored for at least 2 weeks at )80 °C. Fractions (200 lL) were collected into 100 lL column buffer containing 30% glycerol and 2 m M ATP. The fractions were assayed for thymidine kinase activity under standard assay conditions with 100 l M dThd. The molecular markers (vertical bars) are (from left to right): b-amylase (200 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa) and cytochrome c (12.4 kDa). V e is the elution volume. The standard variation for V e ⁄ V 0 of the marker proteins was below 2% (CV) for more than 20 independent experiments. B. Munch-Petersen Enzymatic regulation of human TK1 FEBS Journal 276 (2009) 571–580 ª 2008 The Author Journal compilation ª 2008 FEBS 573 However, that the presence of MgCl 2 is insignificant can be seen in Fig. 2A, where hTK1 elutes as a tetra- mer whether NaPPP is present with or without MgCl 2 (Fig. 1H). Figure 2B,C shows that TK1 also elutes as a tetramer in the presence of NaPP, whereas the elution profile with sodium orthophos- phate (NaP) indicates a dimer of approximately 48.5 kDa with a shoulder at approximately 100 kDa. In all of these elutions, the same amount of hTK1 was applied (10 ng) and recovery of activities was approximately 20–40%. The lower activity seen in the elution with AMP (Fig. 1G) is due to the inhibi- tory effect of AMP in the assay. The average mass of the eight tetrameric hTK1s was estimated as 103.7 ± 3.2 (SEM) kDa (Table 1). Are the oligomerization pattern and kinetics related? The kinetics of hTK1 is complex and deviates from hyperbolic kinetics, with apparent negative co-oper- ativity and a K 0.5 (substrate concentration at half-max- imal velocity) of approximately 15 lm [12]. However, when hTK1 was incubated with ATP prior to the assay, it showed hyperbolic kinetics with a K m of approximately 0.5 lm. Both enzyme forms have the same V max , meaning that the catalytic efficiency of ATP-incubated hTK1 is approximately 30-fold higher than that of non-incubated hTK1. The two TK1 forms can therefore be referred to as the high- and low- efficiency forms. To explain the apparent negative co-operativity, a model has been proposed whereby the dimer has high K m and the tetramer has low K m , and the ratio between the two forms depends on the dThd concentration [24]. According to this model, the simul- taneous presence in the assay of the two forms will result in the apparent negative co-operative behavior. To further elucidate this, the relationship between the oligomerization status and the kinetic behaviour was investigated, i.e. whether the tetrameric and dimeric forms in Figs 1 and 2 exhibited low or high catalytic efficiency. Therefore, the various incubated hTK1 forms from Figs 1 and 2 were analyzed for their kinetic behavior with dThd, and the results are pre- sented in Figs 3 and 4. Only in cases where TK1 was incubated prior to the assay with the compounds pro- ducing the dimer, i.e. AMP (Fig. 3F) and NaP (Fig. 4D), did the enzyme exhibit low catalytic effi- ciency like non-incubated TK1 (Fig. 3D), i.e. with apparent negative co-operativity as indicated by con- cave Hofstee plots of v versus v ⁄ s (insets to the kinetic plots), Hill coefficients < 1 and high K 0.5 values (Table 1). All of the tetrameric forms showed approxi- mately hyperbolic Michaelis–Menten kinetics, with low K m values between 0.51 and 0.95 lm [mean tetrameric K m value is 0.73 lm ± 0.05 (SEM); Table 1]. These results clearly show that the high-efficiency hyperbolic kinetics with low K m is associated with the tetrameric form and that the low-efficiency negative co-operativi- ty kinetics with high apparent K m is associated with the dimeric form of TK1. Phosphate donor specificity The results from Figs 1–4 showed that inorganic di- and tripolyphosphates were able to induce tetra- merization and hyperbolic kinetics with low K m values similar to the nucleoside di- and tri-phosphates, and Table 1. Native molecular size and kinetic parameters. Incubation conditions for hTK1 Mass (kDa) K 0.5 (lM) n (Hill constant) Phosphate donor capacity b (%) None 57.5 ± 2.7 a (5) 16.4 ± 1.0 (10) 0.75 ± 0.04 (10) – ATP 115 ± 4.5 (5) 0.51 ± 0.03 (10) 1.04 ± 0.04 (10) 100 GTP 101 0.68 ± 0.015 (3) 0.97 ± 0.04 (3) 37 ± 1 CTP 95.5 0.79 ± 0.029 (3) 0.98 ± 0.01 (3) 18 ± 3 UTP 100 0.64 ± 0.072 (3) 1.02 ± 0.04 (3) 19 ± 0.1 ADP 118 0.66 ± 0.11 (3) 0.99 ± 0.01 (3) 4 ± 0.8 AMP 52.7 21.3 ± 4.33 (3) 0.77 ± 0.01 (3) 0 NaPPP 93 0.95 ± 0.04 (2) 1.31 ± 0.08 (2) 0 NaPPP-MgCl 2 98 0.90 ± 0.07 (2) 0.97 ± 0.08 (2) 0 c NaPP-MgCl 2 103 0.73 ± 0.10 (2) 0.92 ± 0.14 (2) 0 c NaP-MgCl 2 48.9 14.2 ± 2.4 (2) 0.6 ± 0.12 (2) 0 c a Values are means ± SEM, with the number of independent experiments in parentheses. b Phosphate donor capacity as a percentage of the activity with ATP measured under standard assay conditions with 2.5 m M of the respective donor replacing ATP. c Measured with 2.5 m M MgCl 2 in the assay. Enzymatic regulation of human TK1 B. Munch-Petersen 574 FEBS Journal 276 (2009) 571–580 ª 2008 The Author Journal compilation ª 2008 FEBS therefore the potential capacity of these compounds for phosphate transfer was compared to those of the other nucleotides. The results are presented in Table 1 and show that the inorganic polyphosphates are not able to act as phosphate donor. This also shows that phosphate donor capacity and the tetramerization effect are two independent events. Impact of enzyme concentration on the oligomerization of hTK1 The above-described experiments were all performed with the native enzyme purified from human lympho- cytes to a final concentration of approximately 5 lgÆmL )1 [25], and the concentration of the applied enzyme in the gel filtration experiments in Figs 1 and 2 was 50 ngÆmL )1 (10 ng applied). Using recombinant techniques, concentrations of pure hTK1 more than 1000–10 000-fold higher can be obtained, enabling considerably higher concentrations during gel filtra- tion. This may explain the appearance of both dimer and tetramer peaks during gel filtration of non-incu- bated recombinant human TK1 (rhTK1), although the tetramer peak is the smallest [19,20]. In these studies, TK1 was applied at a concentration of approximately 3 lgÆmL )1 . Recently, it was reported that human TK1 elutes exclusively as a tetramer when applied at a concentration range of 0.4– 20 mgÆmL )1 [26]. The authors suggest that the high- level expression of TK1 obtained in their work may influence the oligomerization pattern of the enzyme. However, the more than 100-fold higher concentra- tion used in the experiments by Birringer et al. [26] compared to those used by Berenstein et al. [19] and Frederiksen et al. [20] may also explain the different elution profiles. To further clarify this issue and the effect of enzyme concentration on the oligomerization status, the elution profile of rhTK1 was analyzed under the conditions and at the concentrations outlined in Fig. 5. In Fig. 5A, rhTK1 was applied at a concen- tration of 0.2 mgÆmL )1 . As seen from the elution profile, rhTK1 elutes exclusively as a tetramer at this enzyme concentration, similar to the elution pattern reported by Birringer et al. [26]. This shows that, at high concentrations, TK1 is a tetramer independent of the presence of ATP or phosphate groups. In Fig. 5B, rhTK1 was diluted to 6 lgÆmL )1 immediately before gel filtration. Here, the enzyme eluted as both a dimer and a tetramer, with approximately 40% of the enzyme activity in the tetrameric form. In Fig. 5C, the enzyme was treated as in previous stud- ies [19,20], i.e. diluted to 6 lgÆmL )1 , allowed to stand at 4 °C for 2 h, and then stored at )80 °C for at least 2 weeks before gel filtration. This treatment did not affect the enzyme activity, as the same V max was obtained before and after the treatment. As seen from Fig. 5C, only a minor part of the enzyme is in the tetramer form. This elution profile is very similar to those previously reported by Berenstein et al. and Frederiksen et al. [19,20]. In their gel-filtration Fig. 2. Effect of orthophosphate and di- and tri-polyphosphates on oligomerization of native hTK1. hTK1 was diluted and incubated with 3 m M of the indicated nucleotides or phosphate compound without MgCl 2, injected onto the Superdex 200 column, eluted with column buffer without MgCl 2 containing 2 mM of the respective nucleotide or phosphate compound, and assayed as described for Fig. 1. B. Munch-Petersen Enzymatic regulation of human TK1 FEBS Journal 276 (2009) 571–580 ª 2008 The Author Journal compilation ª 2008 FEBS 575 experiments on recombinant human TK1, Li et al. [13] diluted and treated the enzyme as in Fig. 5C and also found a similar elution profile. Together, these observations show that rhTK1 behaves as a tetramer even in the absence of phosphate groups when applied at concentrations of 200 lgÆmL )1 or higher, Fig. 3. Effect of nucleotides on hTK1 dThd substrate kinetics. Native human TK1 (hTK1) was incubated with 3 m M of the indicated nucleotide for 2 h at 4 °C, and stored for at least 2 weeks at )80 °C. The initial velocity with the indicated dThd concentrations was determined as described in Experimental procedures. Open symbols; incubation with nucleotide. Closed symbols; incubation without nucleotide. Inset, Hofstee plots of the data. Fig.4. Effect of NaP, NaPP and NaPPP on hTK1 dThd substrate kinetics. Native human TK1 (hTK1) was incubated pre-assay with 3m M of the indicated compound with or without MgCl 2 , and the initial velocity with the indicated dThd concentrations was determined as described in Experimental procedures. Inset, Hofstee plots of the data. Enzymatic regulation of human TK1 B. Munch-Petersen 576 FEBS Journal 276 (2009) 571–580 ª 2008 The Author Journal compilation ª 2008 FEBS and a substantial amount of enzyme is still observed as a tetramer, even when diluted to 6 lgÆmL )1 , when gel filtration commences immediately after dilution. Further, the time- and storage-dependent differences in behaviour after dilution to 6 lgÆmL )1 indicate that dissociation of the tetramer to the dimer is a slowly progressing process. This is supported by the kinetic behaviour of the recombinant enzyme as shown in Fig. 5D, where the enzyme was diluted from 0.5 mgÆmL )1 immediately before the kinase assay. Under these conditions, the kinetic behaviour was essentially like that of the tetramer form, exhibiting hyperbolic kinetics with a K m of 0.7 lm. This also indicates slow dissociation of the tetramer form, and may explain why linear progress curves are always obtained with all forms of the enzyme and under all incubation conditions. When rhTLK1 is diluted from high storage concentrations to low assay concentra- tions of 2–3 ngÆmL )1 , which is below the limit for the ATP tetramerization effect, the enzyme would be expected to dissociate to the dimer form with higher K m during the assay, and this would result in non- linear progress curves. However, slow dissociation from tetramer to dimer will result in linear progress curves, as consistently observed with this enzyme. Such a slow dissociation may indicate that hTK1 is a hysteretic enzyme. The finding that the two linked phosphate groups in pyrophosphate are sufficient for formation of the tetramer clearly shows that neither the base nor the sugar plays a role in the oligomerization process. This appears to agree with the structural conditions for ATP binding to human TK1. In the first crystal structure of TK1-type enzymes of human and myco- plasmic origin [14], the feedback inhibitor dTTP was bound in the substrate pocket, similar to the binding of dTTP to the D. melanogaster multi-substrate deoxyribonucleoside kinase [16], despite the funda- mental differences between the two structures. The three phosphate groups bind backwards, and the thy- mine group is buried in a cleft between the a ⁄ b domain and the so-called lasso domain, a domain that is unique to TK1-type enzymes. The same pattern is seen with other TK1 types of bacterial Fig. 5. Effect of concentration of recombinant human TK1 on oligo- merization and kinetics. The column and dilution buffer used and the assay performed are described in Fig. 1. (A) 40 lg was applied at a concentration of 0.6 mgÆmL )1 . (B, C) 1 lg was applied at a concentration of 6 lgÆmL )1 . In (B), the enzyme was diluted immedi- ately before application, whereas in (C), the enzyme was diluted, incubated for 2 h at 4 °C, and stored at )80 °C for more than 2 weeks. (D) dThd substrate kinetics with recombinant human TK1 (0.1 ng in 50 lL assay reaction volume) diluted from 0.6 mgÆmL )1 to 0.01 lgÆmL )1 immediately before assay. Inset, Hofstee plot of the data. B. Munch-Petersen Enzymatic regulation of human TK1 FEBS Journal 276 (2009) 571–580 ª 2008 The Author Journal compilation ª 2008 FEBS 577 origin [21,22]. In a recent study, the bi-substrate inhi- bitor P1-(5¢-adenosyl)P4-[5¢-(2¢-deoxy-thymidyl)] tetra- phosphate (AP4dT) was crystallized together with hTK1 and TmTK [17,23]. In both structures, thy- mine and the three phosphates were bound in the lasso motif, essentially as for dTTP in the previous structures. The authors conclude that the fourth phosphate, which is analogous to the a-phosphate in ATP, is observed in both structures, whereas electron density is obtained only with the adenine group in the TmTK structure [23]. Moreover, with the ana- logue bound, the TmTK structure appears more open than the hTK1 structure. This indicates that the adenine group in the hTK1 structure makes only a few, if any, contacts with the enzyme. It may also explain at least partly why the kinetic and oligomeric effects can be exhibited by only two phosphate groups, which probably are analogous to the a and b phosphate groups in the nucleotide ADP. On the other hand, the large difference in phosphate donor capacity, only 4% with ADP and no activity with NaPPP and NaPP, indicates that the base part of the phosphate donor must play an essential role in the catalytic process. The physiological TK1 concentration is estimated to increase from approximately 0.03–0.09 lgÆmL )1 (1.2–3.6 nm)inG 0 and G 1 cells to approximately 4–6 lgÆmL )1 (160–240 nm) in peak S-phase cells [12], assuming equal distribution throughout the cytoplasm. This indicates that, in G 1 ⁄ early S phase, TK1 will be in the dimer form, irrespective of the cellular ATP con- centration, due to the low enzyme concentration. As the TK1 concentration increases during S phase, more and more of the enzyme will be in the tetramer form as previously proposed [12]. Further, as shown here, high-efficiency kinetics with low K m values is exclu- sively displayed by the tetramer forms, and low- efficiency kinetics with high K m values is displayed by the dimer forms. These observations strengthen the previous hypothesis that the dimer ⁄ tetramer inter- change of TK1 with low ⁄ high catalytic efficiency is a fine-tuning mechanism that may serve to provide a bal- anced supply of dTTP throughout the cell cycle, adjusted to the need for DNA synthesis [12,13,24]. As dTTP is a key regulator of ribonucleotide reductase, higher dTTP concentrations will result in unbalanced dNTP pools, which are known to be mutagenic [27– 29]. In the light of these effects, the complex regulatory and structural properties of hTK1 may be important for maintaining a balanced supply of the DNA precur- sor. This underlines the importance of elucidating the molecular and structural background of the enzymatic and catalytic properties of human thymidine kinase. Experimental procedures Superdex 12, Glutathione–Sepharose, pGEX-2T vector, thrombin, [methyl- 3 H]dThd (25 CiÆmmol )1 ) and the Esc- herichia coli strains XL Gold and BL21 were purchased from Amersham Biosciences (now part of GE Healthcare Bio-Sciences, Hillerod, Denmark). Strains XL Gold and BL21 were used to propagate and express, respectively, the recombinant thymidine kinase. Chaps was purchased from Roche A/S (Copenhagen, Denmark). Triton X-100, dithiotreitol, non-radioactive nucleosides and molecular mass markers were purchased from Sigma-Aldrich (Copen- hagen, Denmark). Materials for cloning, PCR, DNA seq- uencing and assays were standard commercially available products. Enzyme preparation Native human TK1 (hTK1) was purified from human lymphocytes as previously described [25]. Briefly, superna- tant from streptomycin-precipitated crude cellular homoge- nate was precipitated with ammonium sulfate, desalted on Sephadex G-25, separated from other deoxynucleoside kinases by ion-exchange chromatography on a DEAE column, and further purified by affinity chromatography on a 3¢-dTMP Sepharose column. dThd from the affinity chromatography step was removed, and hTK1 was con- centrated on a carboxymethyl-Sepharose column as described previously [12]. Recombinant human TK1 (rhTK1) was expressed using the pGEX-2T-LyTK1 val106 vector [19], the bacteria were harvested after induction with 0.1 mm isopropyl-1-thio- b-d-galactopyranoside for 6 h at 25 °C, rhTK1 was purified by glutathione–Sepharose chromatography, and the thrombin cleavage fractions were further purified by carboxymethyl chromatography as previously des- cribed [19]. Pre-assay incubation and storage of enzymes Both native and recombinant hTK1 were diluted to a con- centration of 6 lgÆmL )1 in Superdex column buffer (50 mm imidazole ⁄ HCl pH 7.5, 5 mm MgCl 2 , 0.1 m KCl, 2 mm Chaps and 5 mm dithiothreitol), incubated with or without 3mm of the respective nucleotide or phosphate compound for 2 h at 4 °C, and stored for at least 2 weeks at )80 °C before use for kinetic and molecular mass analyses. The activity at saturating conditions was similar before and after dilution, incubation and storage. Native molecular size The apparent molecular size was determined by gel filtra- tion on a Superdex 12 (10 · 300 mm) column connected to a Gradifrac automatic sampler (Amersham Biosciences) as Enzymatic regulation of human TK1 B. Munch-Petersen 578 FEBS Journal 276 (2009) 571–580 ª 2008 The Author Journal compilation ª 2008 FEBS described previously [19]. The column was pre-equilibrated in column buffer (50 mm imidazole ⁄ HCl pH 7.5, 5 mm MgCl 2 , 0.1 m KCl, 2 mm Chaps and 5 mm dithiothreitol) containing two milimolar of the respective nucleotide or phosphate compounds. In each experiment, 0.2 mL enzyme dilution containing 0.1 mg Blue Dextran 2000 (Sigma- Aldrich) was applied. Blue dextran was used as an internal standard for determination of the void volume V 0 of the column. This value was used for calculation of V e ⁄ V 0 . The column was standardized using the following marker pro- teins: b-amylase, 200 kDa; BSA, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa; cytochrome c, 12.4 kDa. This approach ensures high reproducibility in determination of the molecular mass, as the standard varia- tion in V e ⁄ V 0 for the markers was less than 2% (coefficient of variation) from 20 separate marker elution profiles. Fractions (200 lL) were collected into 100 lL column buf- fer containing 30% glycerol and 2 mm ATP for preserva- tion of enzyme activity. The fractions were assayed for thymidine kinase activity under standard assay conditions with 100 lm dThd. Thymidine kinase assay Thymidine kinase activity was assayed by measuring ini- tial velocities using the DE-81 filter paper method as described previously [12,19]. Standard assay conditions were 50 mm Tris ⁄ HCl pH 7.5, 2.5 mm ATP, 2.5 mm MgCl 2 ,10mm dithiothreitol, 0.5 mm Chaps, 3 mgÆmL )1 BSA, 3 mm NaF and the indicated concentration of [methyl- 3 H]dThd in a final volume of 50 lL. The reaction was started by adding approximately 0.1 ng enzyme diluted from 6 lgÆmL )1 in ice-cold enzyme dilution buffer (50 mm Tris ⁄ HCl pH 7.5, 1 mm Chaps, 3 mgÆmL )1 BSA) immediately before the start of the reaction. During the first 15 min of the reaction, four samples of 10 lL each, taken at various time points 3, 6, 9 and 12 min after the start of the reaction, were applied to the DE-81 filters. The filters were washed three times for 5 min each in 5mm ammonium formate and once for 5 min in water, and the nucleotides were eluted from the DE-81 filters by shaking for 30 min in 0.2 m KCl ⁄ 0.1 m HCl, after which the radioactivity was determined by scintillation counting. Analysis of kinetic data Kinetic data were fitted by non-linear regression analysis to the Michaelis–Menten equation v ¼ V max ½S=ðK m þ½SÞ or the Hill equation v ¼ V max ½S n =ðK 0:5 n þ½S n Þ using prism 5 from GraphPad Software Inc. (La Jolla, CA, USA; http://www.graphpad.com/), where K m is the Michaelis constant and K 0.5 is the substrate concentration where v = 0.5 V max . When n = 1, there is no co-operativity, and K 0.5 = K m . 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