Purification and characterization of wild-type and mutant TK1 type kinases from Caenorhabditis elegans

Article abstract of DOI:10.1080/15257770600894410

Caenorhabditis elegans has a single deoxynucleoside kinase-like gene. The sequence is similar to that of human TK1, but besides accepting thymidine as a substrate, the C. elegans TK1 (CeTK1) also phosphorylates deoxyguanosine. In contrast to human TK1, the CeTK1 exclusively exists as a dimer with a molecular mass of ～60 kDa, even if incubated with ATP. Incubation with ATP induces a transition into a more active enzyme with a higher k_cat but unchanged K_m. This activation only occurs at an enzyme concentration in the incubation buffer of 0.5 μg/ml (8.42 nM) or higher. C-terminal deletion of the enzyme results in lower catalytic efficiency and stability. Copyright Taylor & Francis Group, LLC.

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Purification and Characterization of
Wild-Type and Mutant Tk1 Type Kinases
From Caenorhabditis Elegans
a
a
T. Skovgaard & B. Munch-Petersen
a
Department of Life Sciences and Chemistry , Roskilde University ,
Roskilde, Denmark
Published online: 22 Nov 2006.
To cite this article: T. Skovgaard & B. Munch-Petersen (2006) Purification and Characterization of
Wild-Type and Mutant Tk1 Type Kinases From Caenorhabditis Elegans , Nucleosides, Nucleotides and
Nucleic Acids, 25:9-11, 1165-1169, DOI: 10.1080/15257770600894410
To link to this article: http://dx.doi.org/10.1080/15257770600894410
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Nucleosides, Nucleotides, and Nucleic Acids, 25:1165–1169, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600894410
PURIFICATION AND CHARACTERIZATION OF WILD-TYPE AND
MUTANT TK1 TYPE KINASES FROM CAENORHABDITIS ELEGANS
T. Skovgaard and B. Munch-Petersen
Chemistry, Roskilde University, Roskilde, Denmark
2
Department of Life Sciences and
2
Caenorhabditis elegans has a single deoxynucleoside kinase-like gene. The sequence is similar
to that of human TK1, but besides accepting thymidine as a substrate, the C. elegans TK1 (CeTK1)
also phosphorylates deoxyguanosine. In contrast to human TK1, the CeTK1 exclusively exists as
a dimer with a molecular mass of ∼60 kDa, even if incubated with ATP. Incubation with ATP
m
induces a transition into a more active enzyme with a higher kcat but unchanged K . This activation
only occurs at an enzyme concentration in the incubation buffer of 0.5 µg/ml (8.42 nM) or higher.
C-terminal deletion of the enzyme results in lower catalytic efficiency and stability.
Keywords Thymidine kinase; TK1; Caenorhabditis elegans; ATP activation; Characteriza-
tion; Enzyme kinetics
INTRODUCTION
Caenorhabditis elegans thymidine kinase (CeTK1) is a TK1 type deoxynu-
cleoside kinase that catalyzes the transfer of γ -phosphate from ATP to
ꢁ
the 5 -OH of thymidine and deoxyguanosine to form dTMP and dGMP,
respectively. In humans and other mammalians TK1 is a key enzyme in the
salvage of dTTP for DNA synthesis.
Deoxynucleoside kinases are also important for activation of antiviral
and anticancer drugs such as the nucleoside analog AZT used for HIV
treatment. A thymidine kinase from Herpes Simplex virus type 1, HSV1-TK,
[
1,2]
has been used in suicide gene therapy in an attempt to cure cancer.
In
tumor cells expressing the HSV1-TK, the analog is phosphorylated to the
monophosphate level. Subsequently, cellular kinases add 2 more phosphate
groups to form the triphosphate analog, which after incorporation into
We would like to thank Zoran Gojkovic from ZGene for providing the CeTK1-gene and Marianne
Lauridsen and Lene S. Harlow for excellent technical assistance.
Address correspondence to B. Munch-Petersen, Department of Life Sciences and Chemistry,
Roskilde University, DK-4000, Roskilde, Denmark. E-mail: bmp@ruc.dk
1
165

1
166
T. Skovgaard and B. Munch-Petersen
DNA prevents DNA elongation and causes cell cycle arrest or apoptosis.
In order to find a better deoxynucleoside kinase with higher analog
specificities for this purpose, new enzymes are continuously characterized
and new analogs are created.
In the present characterization of CeTK1, the enzyme is compared
with human TK1 (HuTK1) with regard to native size, activation by ATP,
stability and kinetic parameters for thymidine. CeTK1 is truncated in
the C-terminal in order to see how the truncation affects stability and
activity.
MATERIALS AND METHODS
The CeTK1 gene was provided from ZGene (Hoersholm, Denmark) and
cloned into the expression vector pGEX-2T by PCR amplifcation (Primers:
ꢁ
Forward: 5 -CGG CGC CCG GGC ATG GAC ATT GAA GCG GCC AAG AAT
ꢁ
ꢁ
GAG ATG ACT TGC-3 ; Reverse: 5 CCG GAA TTC TTA AGT CCG AGC
TGT GGC CTC CAG AGC 3 ) followed by cleavage and ligation at XmaI and
ꢁ
EcoRI restriction sites. CeTK1 was expressed in E. coli BL21 as a GSH fusion
[
3]
protein and purified by GSH affinity chromatography as described.
The C-terminally truncated mutant was constructed using the
QuickChange site-directed mutagenesis kit from Stratagene (AH Diagnos-
ꢁ
tics, Denmark) (Primers: Forward: 5 -GC AGAGAA TGT TAT GTT CAA
ꢁ
ꢁ
AAG AGC TAA GAA AAA GAT GC-3 ; Reverse: 5 GC ATC TTT TTC TTA
GCT CTT TTG AAC ATA ACA TTC TCT GC-3 . Mutation marked with
ꢁ
grey). The truncated enzyme was expressed and purified as the wild type.
Effect of ATP on the enzymes was examined by incubating 5 µg/ml
(84.2 nM) enzyme for 2 hours on ice in buffer A (50 mM Tris HCL pH
7
.5, 5 mM MgCl , 0.1 M KCl, 2 mM 3-[(3-Cholamidopropyl)-dimethyl-
2
ammonium]-1-propanesulfonate (CHAPS), 10% Glycerol, 5 mM dithiothre-
itol (DTT)) with 2.5 mM ATP. For comparison, an aliquot of enzyme was
incubated in the same buffer without ATP.
Enzyme activities were measured as initial velocities determined by
3
time samples (5, 10, and 15 minutes) with the DE-81 filter pa-
3
[3]
per assay using H labelled substrates.
Reaction mixture of 50 µl
contained 50 mM TRIS-HCl, pH 7.5, 2.5 mM MgCl , 10 mM DTT,
2
0
.5 mM CHAPS, 3 mM NaF, 3 mg/ml BSA, 2.5 mM ATP, and
3
−1
[
methyl- H]-Thymidine (1.8 Ci mmol
Amersham Pharmacia Biotech,
now GE Healthcare Bio-Sciences, Hilleroed, Denmark) and 0.074 nM
enzyme.
The native size was determined by gel filtration on a superdex 200
column as described.[
3]
CeTK1 stability was determined by incubating the enzyme at room
temperature (5 µg/ml, 84.2 nM) in buffer A with or without ATP.

C. elegans Thymidine Kinase 1
RESULTS AND DISCUSSION
1167
The entire genome of C. elegans is sequenced and appears to con-
tain only a single deoxynucleoside kinase-like sequence. Humans have 4
different deoxynucleoside kinases whereas insects, such as the fruit fly
Drosophila melanogaster, only have a single one. However, in contrast to the
human deoxynucleoside kinases, the insect deoxynucleoside kinase can
phosphorylate all 4 naturally occurring deoxynucleosides and differ greatly
[
4–6]
in structure from the TK1-type kinases.
A BLAST search on the CeTK1
protein sequence reveals the closest similarity to HuTK1, with 46% identical
and 63% positive matches. A sequence alignment of HuTK1 and CeTK1
is presented in Figure 1, and it is evident from the alignment that the
two enzymes have high sequence similarity with exception of the N- and
C-terminal regions.
In spite of the high similarity, HuTK1 and CeTK1 differ in respect to
both substrate specificity and ATP-activation pattern: When HuTK1 is incu-
bated with ATP it changes from a dimer form with a mass of ∼50 kDa and
a high K of ∼15 µM into a tetramer of ∼100 kDa with an approximately
m
[
7,8]
2
0– to 30- fold lower K (Table 1).
k
cat
for HuTK1 is in the same range
m
for both the dimer and tetramer. In contrast to this, CeTK1 is a dimer of
60 kDa regardless of ATP incubation, but even so ATP has an effect on
∼
enzyme activity. The ATP incubated CeTK1 has a higher k than the enzyme
cat
incubated without ATP, but Km is unchanged. Hence, ATP activates both
enzymes, but HuTK1 is activated with regard to K and CeTK1 with regard to
m
kcat. In general, the kcat values for HuTK1 and CeTK1 are in the same range,
but for the enzymes not incubated with ATP, the HuTK1 kcat is higher than
FIGURE 1 ClustalX 1.8 alignment of sequences of HuTK1 (GeneBank nr. AAH06484) and CeTK1
∗
(
GeneBank nr. NM 069485). ( Indicates a conserved amino acid.) The gray boxes indicate the position
for C-terminal truncation of the 2 enzymes.

1
168
T. Skovgaard and B. Munch-Petersen
TABLE 1 Kinetic Values with the Substrate Thymidine
HuTK1a
WT (25.5 kDa)
Cꢀ40 (21.1 kDa)
Storage conditions
−ATP
+ATP
−ATP
+ATP
−1
kcat (sec
)
6.3 ± 0.9
16 ± 3.4
6.9 ± 0.2
0.7 ± 0.2
9.5 ± 1.1
1.4 ± 0.6
9.7 ± 0.2
0.6 ± 0.1
Km (µM)
kcat/Km
6
6
6
6
0.4 × 10
9.9 × 10
6.8 × 10
16.2 × 10
CeTK1
WT (29.7 kDa)
Cꢀ56 (23.6 kDa)
Storage conditions
−ATP
+ATP
−ATP
+ATP
−
1 b
kcat (sec
)
5.0 ± 0.1
2.3 ± 0.3
8.8 ± 0.2
2.6 ± 0.3
1.1 ± 0.0
7.4 ± 0.8
3.5 ± 0.2
5.8 ± 0.9
Km (µM)
kcat/Km
6
6
6
6
2.2 × 10
3.4 × 10
0.1 × 10
0.6 × 10
a
Kinetic values for HuTK1 are from.[8]
kcat values were calculated presuming one binding site per subunit using the theoretical MW
b
for the enzyme. +ATP and −ATP denotes whether the enzyme has been pre-incubated in a buffer
with or without 2.5 mM ATP/MgCl2.
the CeTK1 k , and for the ATP incubated enzymes, CeTK1 k is slightly
cat
cat
higher than the HuTK1 kcat.
Due to a high K value and a low solubility of deoxyguanosine accurate
m
kinetic values for phosphorylation of this substrate could not be achieved.
The ATP-activation of CeTK1 depends on enzyme concentration in
the incubation buffer. At concentrations of 0.1 µg/ml (1.68 nM) and
lower there is no transition to the more active +ATP form of the enzyme,
whereas at 0.5 µg/ml (8.42 nM) and higher concentrations the transition
to the higher kcat form takes place. The low enzyme concentration in the
assay (0.074 nM) explains our linear progression curves and absence of
transition during the kinase assay although there is 2.5 mM ATP in the assay
mixture.
CeTK1 is remarkably stable compared to the human TK1. When incu-
bated in TRIS buffer, the half-life of CeTK1 is 222 hours whereas the half-life
of HuTK1 incubated in phosphate buffer (50 mM K-phosphate with 0.5 mM
CHAPS) is only 58 minutes.[ The half-lifes are not directly comparable due
to the different incubation buffers, but since HuTK1 has a lower stability
in TRIS buffer than in phosphate buffer (B. Munch-Petersen, personal
communication) the stability of CeTK1 is more than 540 fold higher than
the stability of HuTK1. However, where deletion of the C-terminal of the
HuTK1 increases the stability,[ similar deletion of the C-terminal of CeTK1
has a destabilizing effect. Thus, the half-life for HuTK1 deleted by 40 amino
8]
8]
[
8]
acids in the C-terminal is 256 minutes, which is 32 = fold higher than
the undeleted enzyme. Deletion of the corresponding amino acids (the
position of deletion is marked on Figure 1) in CeTK1 (CeTK1-Cꢀ56) results
in a 1.4-fold decrease in half-time from 222 hours to 156 hours. Further,
the C-terminal deletion in the human kinase increases the catalytic activity,

C. elegans Thymidine Kinase 1
1169
whereas the C-terminal deletion of CeTK1 decreases the catalytic activity. In
general, CeTK1 is a remarkably stable enzyme both with and without the
◦
C-terminal. During more than two weeks of storage at 4 C it maintains full
activity.
In summary, the results presented here show that in contrast to the
C-terminal of HuTK1 that decreases the stability and enzymatic activity, the
C-terminal of CeTK1 maintains stability and enzymatic activity.
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