Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties

Article abstract of DOI:10.1080/15257770600894436

Thymidine kinase (TK1) is a key enzyme in the salvage pathway of nucleotide metabolism and catalyzes the first rate-limiting step in the synthesis of dTTP, transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5′-hydroxyl group of thymidine, thus forming dTMP. TK1 is cytosolic and its activity fluctuates during cell cycle coinciding with the DNA synthesis rate and disappears during mitosis. This fluctuation is important for providing a balanced supply of dTTP for DNA replication. The cell cycle specific activity of TK1 is regulated at the transcriptional level, but posttranslational mechanisms seem to play an important role for the level of functional TK1 protein as well. Thus, the C-terminal of TK1 is known to be essential for the specific degradation of the enzyme at the G2/M phase. In this work, we have studied the effect of deletion of the C-terminal 20, 40, and 44 amino acids of TK1 on in vitro stability, oligomerization, and enzyme kinetics. We found that deletion of the C-terminal fold markedly increased the stability as well as the catalytic activity. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770600894436

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Effect of C-Terminal of Human Cytosolic
Thymidine Kinase (TK1) on in Vitro
Stability and Enzymatic Properties
C. Zhu a , L. S. Harlow a , D. Berenstein a , S. Munch-Petersen a & B.
Munch-Petersen a
a Department of Life Sciences and Chemistry , Roskilde University ,
Roskilde, Denmark
Published online: 22 Nov 2006.
To cite this article: C. Zhu , L. S. Harlow , D. Berenstein , S. Munch-Petersen & B. Munch-Petersen
(2006) Effect of C-Terminal of Human Cytosolic Thymidine Kinase (TK1) on in Vitro Stability and
Enzymatic Properties, Nucleosides, Nucleotides and Nucleic Acids, 25:9-11, 1185-1188, DOI:
10.1080/15257770600894436
To link to this article: http://dx.doi.org/10.1080/15257770600894436
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Nucleosides, Nucleotides, and Nucleic Acids, 25:1185–1188, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600894436
EFFECT OF C-TERMINAL OF HUMAN CYTOSOLIC THYMIDINE
KINASE (TK1) ON IN VITRO STABILITY AND ENZYMATIC
PROPERTIES
C. Zhu, L. S. Harlow, D. Berenstein, S. Munch-Petersen, and
B. Munch-Petersen 2 Department of Life Sciences and Chemistry, Roskilde University,
Roskilde, Denmark
Thymidine kinase (TK1) is a key enzyme in the salvage pathway of nucleotide metabolism and
2
catalyzes the first rate-limiting step in the synthesis of dTTP, transfer of a gamma-phosphate group
from a nucleoside triphosphate to the 5ꢁ-hydroxyl group of thymidine, thus forming dTMP. TK1 is
cytosolic and its activity fluctuates during cell cycle coinciding with the DNA synthesis rate and
disappears during mitosis. This fluctuation is important for providing a balanced supply of dTTP
for DNA replication.
The cell cycle specific activity of TK1 is regulated at the transcriptional level, but posttransla-
tional mechanisms seem to play an important role for the level of functional TK1 protein as well.
Thus, the C-terminal of TK1 is known to be essential for the specific degradation of the enzyme at
the G2/M phase.
In this work, we have studied the effect of deletion of the C-terminal 20, 40, and 44 amino
acids of TK1 on in vitro stability, oligomerization, and enzyme kinetics. We found that deletion of
the C-terminal fold markedly increased the stability as well as the catalytic activity.
Keywords Thymidine kinase 1; TK1; Cell cycle regulation; C-terminal; Stability
INTRODUCTION
Cytosolic thymidine kinase 1, TK1 (E.C.2.7.1.21) is a key enzyme in the
salvage of thymine nucleotides, catalyzing the first phosphorylation step of
thymidine to TMP which thereafter is quickly phosphorylated to TDP and
TTP. TK1 activity is cell cycle regulated, being low or absent in nondividing
cells, increases during G1/S phase in close correlation to the increase
in DNA synthesis and disappears rapidly in G2/M phase due to specific
We are indebted to Marianne Lauridsen for her excellent technical assistance. The work was
supported by grants from the Danish Research Council.
Address correspondence to B. Munch-Petersen, Department of Life Sciences and Chemistry,
Roskilde University, DK-4000 Roskilde, Denmark. E-mail: bmp@ruc.dk
1185

1186
C. Zhu et al.
proteolytic degradation by the ubiquitin-dependent proteasomal pathway
which recognizes a specific KEN box motif in the C-terminal end of TK1.[1]
Deletion of the 40 C-terminal amino acids of TK1 delayed the G2/M phase
specific degradation.[2]
We previously have found another regulatory mechanism operating at
the enzymatic level, where TK1 can occur in two reversibly interchangeable
forms.[3,4] At low TK1 concentrations, the enzyme is a low activity dimer
(Km about 15 µM). At higher concentrations (>0.2 µg ml−1) and in the
presence of ATP, TK1 occurs as a high activity tetramer (Km about 0.5 µM).
Both enzyme forms have a Vmax about 18–20 µmol min−1 mg−1. We believe it
is very likely that this enzymatic regulation confers a fine-tuning mechanism
of the regulation of TK1 activity during the cell cycle.
The present work was undertaken to investigate the C-terminal amino
acids effect on the enzymatic regulation of TK1, if any.
MATERIALS AND METHODS
TK1 mutants. C-terminal truncated TK1 mutants lacking 20, 40, and
44 C-terminal amino acids (TK1 Cꢀ20, TK1 Cꢀ40, TK1 Cꢀ44) were
constructed from the plasmid pGEX-2T-LyTK1val106 by inserting stop codons
at the desired sites using the QuickChange site-directed mutagenesis kit
(Stratagene, AH Diagnostics, Denmark).
Recombinant enzyme expression and purification. TK1 enzymes were ex-
pressed in E.coli from using the glutathione S-transferase (GST) fu-
sion system (Amersham pharmacia Biothech) and purified in two steps
on glutathione-sepharose 4B (Amersham Pharmacia Biotech) and CM-
sepharose, as previously described.[4] According to SDS PAGE, the purity
was higher than 98%.
Enzyme assay. TK1 activity was assayed as initial velocities by the DE-81
filter paper method as described.[3] The standard reaction mixture
contained 50 mM TRIS-HCl, pH 8.0, 2.5 mM MgCl2, 10 mM DTT,
0.5 mM CHAPS, 3 mg/ml BSA, 2.5 mM ATP and [methyl-3H]-Thymidine
(66.6 GBq/mmol calculate GBq) Amersham Pharmacia Biotech) in a total
volume of 50 µL.
TK1 stability. TK1 (25 ng mL−1) was incubated in 50 mM K-phosphate
buffer with 0.5 mM CHAPS at 20◦C for 5 hours. Time samples of 10 µL
were taken after 0, 10, 20, 45, 60, 90, 120, 180, 240, and 300 minutes of
incubation and added to the standard TK assay reaction mixture with 10 µM
[methyl-3H]-Thymidine (66.6 GBq mmol−1).
Molecular size. Subunit molecular size is determined by SDS PAGE. Native
molecular size is determined by gelfiltration on a Superdex 200 column (10
× 300 mm) as described.[5] The elution buffer is 50 mM imidazole/HCl pH
7.5, 5 mM MgCl2, 0.1 M KCl, 2 mM CHAPS, 5 mM dithiothreitol. 0.7 µg pure

TK-1 C-Terminal Properties
1187
TABLE 1 Enzymatic Properties of Untruncated and C-Terminal Truncated TK1
TK1 WT
TK1 Cꢀ40
Storage conditions
−ATP
+ATP
−ATP
+ATP
a
kcat (sec−1
Km (µM)
)
6.3 0.9
16 3.4
60
6.9 0.2
0.7 0.2
100
9.5 1.1
1.4 0.6
60
9.7 0.2
0.6 0.1
90
Molecular size (kD)b
akcat values (with SDs based on 2 experiments) were calculated presuming one binding
site per subunit using the theoretical MW of TK1. bThe molecular size was determined as
described in MATERIALS AND METHODS.
TK1 was applied. The +ATP samples contained 2.5 mM ATP and 2.5 mM
ATP was added to the elution buffer.
RESULTS AND DISCUSSION
We have examined the effect of deletion of the C-terminal amino acids
on the enzymatic properties and stability of human recombinant TK1.
Deletion of the 40 C-terminal amino-acids of TK1 had minor impact on
the enzymatic properties (Table 1), since the difference between the +
and −ATP forms is considerably less pronounced compared to the WT
TK1. However, when studying the ability to occur as dimer in absence and
tetramer in presence of ATP, the truncated form behaved essentially as the
untruncated (Table 1).
In contrast to the minor effect of truncation on kinetics and native
molecular size, the impact on the stability was pronounced. As seen from
Table 2, the half-life of TK1 increases with increasing truncation. Deletion
of 44 amino acids increased the half life 6-fold. However, deletion of these
additional four amino acids had a marked impact on kcat that decreased
almost 4-fold compared to the TK1Cꢀ40 which may be due to the close
vicinity to the highly conserved Zn-binding domain.[6]
Clearly, the C-terminus confers instability to TK1 and may have the
function to introduce unfolding of the enzyme to expose the KEN box for
cell-cycle specific degradation.
TABLE 2 Stability and kcat Values for TK1 WT and C-Terminal Deletion Mutants
TK1 WT
TK1 Cꢀ20
TK1 Cꢀ40
TK1 Cꢀ44
t1/2 (min)a
kcat (sec−1
83
6.3 0.9
7
78
3.7 0.7
6
335 24
9.5 1.19
500 70
2.5 0.3
)
aThe time of incubation at 20◦C, where 50% activity is lost. The incubation
buffer was 50 mM potassium phosphate pH 7.5 with 0.5 mM CHAPS.

1188
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C. Zhu et al.
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2538–2546.
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