Tài liệu Báo cáo khoa học: Reversible tetramerization of human TK1 to the high catalytic efficient form is induced by pyrophosphate, in addition to tripolyphosphates, or high enzyme concentration ppt

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:
Website: />(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; 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
.
Acknowledgements
This work was supported by grants from the Danish
Research council and the Novo Nordic Research
Council. The skilful technical assistance of Marianne
Lauridsen is gratefully acknowledged.
References
1 Malinsky J, Koberna K, Stanek D, Masata M, Votruba
I & Raska I (2001) The supply of exogenous deoxyribo-
nucleotides accelerates the speed of the replication fork

in early S-phase. J Cell Sci 114, 747–750.
2 Chabes A & Stillman B (2007) Constitutively high
dNTP concentration inhibits cell cycle progression and
the DNA damage checkpoint in yeast Saccharomyces
cerevisiae. Proc Natl Acad Sci USA 104, 1183–1188.
3 Koc A, Wheeler LJ, Mathews CK & Merrill GF (2004)
Hydroxyurea arrests DNA replication by a mechanism
that preserves basal dNTP pools. J Biol Chem 279,
223–230.
4 Munch-Petersen B, Tyrsted G & Dupont B (1973) The
deoxyribonucleoside 5¢-triphosphate (dATP and dTTP)
pools in phytohemagglutinin-stimulated and non-stimu-
lated human lymphocytes. Exp Cell Res 79, 249–256.
5 Tyrsted G (1975) The pool size of deoxyguanosine
5¢-triphosphate and deoxycytidine 5¢-triphosphate in
phytohemagglutinin-stimulated and non-stimulated
human lymphocytes. Exp Cell Res 91, 429–440.
6 Chabes A & Thelander L (2003) DNA building blocks
at the foundation of better survival. Cell Cycle 2, 171–
173.
7 Snyder RD (1988) Consequences of depletion of cellular
deoxyribonucleoside triphosphate pools on the exci-
sion–repair process in cultured human fibroblasts.
Mutat Res 200, 193–199.
8 Reichard P (1988) Interactions between deoxyribonucle-
otide and DNA synthesis. Annu Rev Biochem 57, 349–
374.
9 Pontarin G, Ferraro P, Valentino ML, Hirano M, Rei-
chard P & Bianchi V (2006) Mitochondrial DNA deple-
tion and thymidine phosphate pool dynamics in a

cellular model of mitochondrial neurogastrointestinal
encephalomyopathy. J Biol Chem 281, 22720–22728.
10 Eriksson S, Munch-Petersen B, Johansson K & Ekl-
und H (2002) Structure and function of cellular
deoxyribonucleoside kinases. Cell Mol Life Sci 59,
1327–1346.
11 Ke PY & Chang ZF (2004) Mitotic degradation of
human thymidine kinase 1 is dependent on the ana-
phase-promoting complex ⁄ cyclosome–CDH1-mediated
pathway. Mol Cell Biol 24, 514–526.
12 Munch-Petersen B, Tyrsted G & Cloos L (1993)
Reversible ATP-dependent transition between two
forms of human cytosolic thymidine kinase with
B. Munch-Petersen Enzymatic regulation of human TK1
FEBS Journal 276 (2009) 571–580 ª 2008 The Author Journal compilation ª 2008 FEBS 579
different enzymatic properties. J Biol Chem 268, 15621–
15625.
13 Li CL, Lu CY, Ke PY & Chang ZF (2004) Perturba-
tion of ATP-induced tetramerization of human cytosolic
thymidine kinase by substitution of serine-13 with
aspartic acid at the mitotic phosphorylation site. Bio-
chem Biophys Res Commun, 313, 587–593.
14 Welin M, Kosinska U, Mikkelsen NE, Carnrot C, Zhu
C, Wang L, Eriksson S, Munch-Petersen B & Eklund H
(2004) Structures of thymidine kinase 1 of human and
mycoplasmic origin. Proc Natl Acad Sci USA 101,
17970–17975.
15 Sabini E, Ort S, Monnerjahn C, Konrad M & Lavie A
(2003) Structure of human dCK suggests strategies to
improve anticancer and antiviral therapy. Nat Struct

Biol 10, 513–519.
16 Johansson K, Ramaswamy S, Ljungcrantz C, Knecht
W, Piskur J, Munch-Petersen B, Eriksson S & Eklund
H (2001) Structural basis for substrate specificities of
cellular deoxyribonucleoside kinases. Nat Struct Biol 8,
616–620.
17 Segura-Pena D, Lichter J, Trani M, Konrad M, Lavie
A & Lutz S (2007) Quaternary structure change as a
mechanism for the regulation of thymidine kinase 1-like
enzymes. Structure 15, 1555–1566.
18 Bradshaw HD Jr & Deininger PL (1984) Human thymi-
dine kinase gene: molecular cloning and nucleotide
sequence of a cDNA expressible in mammalian cells.
Mol Cell Biol 4, 2316–2320.
19 Berenstein D, Christensen JF, Kristensen T, Hofbauer
R & Munch-Petersen B (2000) Valine, not methionine,
is amino acid 106 in human cytosolic thymidine kinase
(TK1). Impact on oligomerization, stability and kinetic
properties. J Biol Chem 275, 32187–32192.
20 Frederiksen H, Berenstein D & Munch-Petersen B
(2004) Effect of valine 106 on structure–function rela-
tion of cytosolic human thymidine kinase. Kinetic prop-
erties and oligomerization pattern of nine substitution
mutants of V106. Eur J Biochem 271, 2248–2256.
21 Birringer MS, Claus MT, Folkers G, Kloer DP, Schulz
GE & Scapozza L (2005) Structure of a type II thymi-
dine kinase with bound dTTP. FEBS Lett 579, 1376–
1382.
22 Kosinska U, Carnrot C, Eriksson S, Wang L & Eklund
H (2005) Structure of the substrate complex of thymi-

dine kinase from Ureaplasma urealyticum and investiga-
tions of possible drug targets for the enzyme. FEBS J
272, 6365–6372.
23 Segura-Pena D, Lutz S, Monnerjahn C, Konrad M &
Lavie A (2007) Binding of ATP to TK1-like enzymes is
associated with a conformational change in the quater-
nary structure. J Mol Biol 369, 129–141.
24 Munch-Petersen B, Cloos L, Jensen HK & Tyrsted G
(1995) Human thymidine kinase 1. Regulation in
normal and malignant cells. Adv Enzyme Regul 35 ,
69–89.
25 Munch-Petersen B, Cloos L, Tyrsted G & Eriksson S
(1991) Diverging substrate specificity of pure human
thymidine kinases 1 and 2 against antiviral dideoxy-
nucleosides. J Biol Chem 266, 9032–9038.
26 Birringer MS, Perozzo R, Kut E, Stillhart C, Surber W,
Scapozza L & Folkers G (2006) High-level expression
and purification of human thymidine kinase 1: quater-
nary structure, stability, and kinetics. Protein Expr Purif
47, 506–515.
27 Kunz BA, Kohalmi SE, Kunkel TA, Mathews CK,
Mcintosh EM & Reidy JA (1994) International Com-
mission for Protection Against Environmental Muta-
gens and Carcinogens. Deoxyribonucleoside
triphosphate levels: a critical factor in the maintenance
of genetic stability. Mutat Res 318, 1–64.
28 Kunz BA & Kohalmi SE (1991) Modulation of muta-
genesis by deoxyribonucleotide levels. Annu Rev Genet
25, 339–359.
29 Ke PY, Kuo YY, Hu CM & Chang ZF (2005) Control

of dTTP pool size by anaphase promoting complex
⁄
cyclosome is essential for the maintenance of genetic
stability. Genes Dev 19, 1920–1933.
Enzymatic regulation of human TK1 B. Munch-Petersen
580 FEBS Journal 276 (2009) 571–580 ª 2008 The Author Journal compilation ª 2008 FEBS