From the Department of Veterinary Medical Chemistry,
Swedish University of Agricultural Sciences, The Biomedical Centre,
P. O. Box 575, SE-751 23 Uppsala, Sweden and the ¶ Metabolic
Disease Unit, Shaare Zedek Medical Center, P. O. Box 3235, Jerusalem 91031, Israel
Received for publication, June 20, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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Thymidine kinase 2 (TK2) is a mitochondrial (mt)
pyrimidine deoxynucleoside salvage enzyme involved in mtDNA precursor
synthesis. The full-length human TK2 cDNA was cloned and sequenced.
A discrepancy at amino acid 37 within the mt leader sequence in the DNA
compared with the determined peptide sequence was found. Two mutations in the human TK2 gene, His-121 to Asn and Ile-212 to Asn, were recently
described in patients with severe mtDNA depletion myopathy (Saada, A.,
Shaag, A., Mandel, H., Nevo, Y., Eriksson, S., and Elpeleg, O. (2001)
Nat. Genet. 29, 342-344). The same mutations in TK2 were
introduced, and the mutant enzymes, prepared in recombinant form, were
shown to have similar subunit structure to wild type TK2. The I212N
mutant showed less than 1% activity as compared with wild type TK2
with all deoxynucleosides. The H121N mutant enzyme had normal
Km values for thymidine (dThd) and deoxycytidine (dCyd), 6 and 11 µM, respectively, but 2- and 3-fold lower Vmax values as compared with
wild type TK2 and markedly increased Km values for
ATP, leading to decreased enzyme efficiency. Competition experiments
revealed that dCyd and dThd interacted differently with the H121N
mutant as compared with the wild type enzyme. The consequences of the
two point mutations of TK2 and the role of TK2 in mt disorders are discussed.
Mitochondrial DNA
(mtDNA)1 replication is not
cell cycle-regulated; therefore, a constant supply of deoxynucleoside
triphosphates (dNTPs) is required. Two mitochondrial membranes separate
these dNTP pools from the cytosolic/nuclear dNTP pools, and the mt
dNTPs are either imported from cytosol or synthesized in
situ in mitochondria by salvage enzymes. In proliferating cells
the biosynthesis of dNTPs occurs via de novo synthesis, but
in resting cells or terminally differentiated cells salvage of
pre-existing deoxynucleosides is essential for providing the dNTPs for
nuclear DNA repair and mtDNA synthesis (1-3).
In mammalian cells the first step in the salvage of deoxynucleosides is
carried out by thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK)
in the cytosol and thymidine kinase 2 (TK2) and deoxyguanosine kinase
(dGK) in the mitochondria. Both TK1 and TK2 use thymidine (dThd)
and deoxyuridine as substrates, while TK2 also phosphorylates deoxycytidine (dCyd). The expression of TK1 is cell cycle-regulated with the highest level in S phase cells and very low or no activity in
resting cells. In contrast TK2 is constitutively expressed at low level
in all tissues. dCK is able to phosphorylate dCyd, deoxyadenosine
(dAdo), and deoxyguanosine (dGuo) and is mainly expressed in lymphoid
tissues. Similar to dCK, dGK phosphorylates dGuo, dAdo, and to some
extend dCyd and is constitutively expressed in all tissues (1-3).
Abnormal mt dNTP synthesis has recently been associated with inherited
mitochondrial DNA depletion disorders such as mitochondrial neurogastrointestinal encepalomyopathy and external opthalomoplegia due
to deficiencies in thymidine phosphorylase and the mt ADP/ATP translocator protein, respectively (4-6). These enzymes, like all
other mtDNA replication factors/enzymes, are coded by nuclear genes,
and these types of autosomal disorders are defects in the cross-talk
between the mitochondrial and nuclear genomes, manifested as multiple
deletions and depletion of mtDNA (5).
Very recently individuals lacking dGK activity were described, and they
showed severe hepatocerebral symptoms due to mtDNA depletion in the
affected tissues (7). The patients had early onset of progressive liver
failure, lactic acidosis and neurological abnormalities. The molecular
defect was a deletion in the dGK gene leading to truncation of the dGK
protein and total lack of the protein in liver. The lack of dGK
supposedly led to defective synthesis of dATP and dGTP needed for mtDNA
replication (7).
Simultaneously another type of mtDNA depletion disease was described
where two single point mutations in the TK2 gene, giving H121N and
I212N substitutions, were found. The numbering used here is based on
the full-length TK2 sequence, and they were previously numbered as
His-90 and Ile-181 (8). This disease was associated with devastating
mitochondrial myopathy in infancy of patients from four different
families (8). The characterization of the TK2 activity in extracts from
muscle mitochondria from the patients was complicated due to the low
the levels of TK2 activity found in the extracts. Therefore a more
detailed characterization of these two TK2 mutants with purified enzyme
was needed.
In the present study we have used site-directed mutagenesis to
introduce the same mutations in TK2 as those found in the patients and
characterized the recombinant enzymes in kinetic experiments with dThd,
dCyd, and ATP. Inhibition studies with the substrates and feedback
inhibitors dCTP and dTTP were also performed. A mechanism for the
observed mtDNA depletion is presented, and a characterization of the
full-length human TK2 cDNA, including the mt leader sequence, is also presented.
Materials--
[methyl-3H]thymidine (25 Ci/mmol) and [5-3H]deoxycytidine (24 Ci/mmol) were
purchased from Amersham Biosciences. Non-radioactive nucleosides
were from Sigma, and all other chemicals were of the highest quality available.
The Cloning and Sequencing of the TK2 Gene--
Human TK2
cDNA (10) was used as probe to screen the human genomic BAC library
(Genome Systems Inc.), and positive clones were identified and
purchased. The TK2 gene was either sequenced directly by using the BAC
DNA or subcloned into the BlueScript vector and then sequenced with
BigdyeTM terminator kit and the Prisma 300 system
(PerkinElmer Life Sciences). Sequencing primers were chosen from exon
sequences. The first exon was identified and used to search the
GenBankTM databases with the BLAST program. Full-length TK2
cDNA clones were identified, and one clone (accession number
AL583655) was obtained from ResGenTM (Invitrogen) and
sequenced. The full-length human TK2 sequence has been deposited in the
GenBank/EMBL/DDBJ databases under the accession number of Y10498.
Mutagenesis--
An N-terminal-truncated TK2 protein, starting
from amino acid number 51 according to the full-length human TK2
sequence presented here, had been characterized earlier, and it showed
identical kinetic properties to those of the native enzyme (10).
Therefore, this form of the enzyme was used as wild type TK2 in this
study. Two complementary oligonucleotides were designed for each
mutant, and the forward oligo sequences were for H121N mutant:
5'-TGGACAGGAATACTCGTCCTC; for the I212N mutant,
5'-CCTGGAAGCAAATCACCATC; bold letters indicate the desired
mutations. The T7 promoter primer was paired with reverse mutant
oligos, and forward mutant oligos were paired with the T7 terminator
primer, and wild type TK2 cDNA, which has been cloned into the
pET-14b vector, was used as template in PCR reactions. The amplified
two PCR fragments were purified and fused together due to their
sequence complementation and used as templates later to amplify the
entire sequence using the T7 promoter primer and the T7
terminator primer. This later PCR product was digested with
NdeI and BamHI and then subcloned into the
pET-14b vector (Novagen, Madison). The mutations were verified by sequencing.
Expression and Purification--
The plasmids that contained the
desired mutations were transformed into BL21 (DE3) pLysS bacteria.
Induction was performed for 2 h in the presence of 0.4 mM
isopropyl-1-thio- Enzyme Assay--
TK2 activity was determined by using
[3H]dThd or [3H]dCyd as substrates as
described previously (10). The standard reaction mixture contains 50 mM Tris/HCl, pH 7.6, 2 mM ATP, 2 mM
MgCl2, 0.5 mg/ml BSA, 5 mM DTT, and 11 µM [3H]-labeled nucleoside and purified
enzyme. One unit is defined as the formation of 1 nmol product (dTMP or
dCMP) per min per mg of protein. For the measurement of the kinetic
constants a wide concentration range of each substrate was used. The
data were analyzed by the Sigma Plot Enzyme Kinetic Module version 1.1 (SPSS Inc).
Gelfiltration Chromatography--
Purified enzymes were analyzed
on a Superdex 200 column using an fast protein liquid chromatography
system (Amersham Biosciences) in a buffer containing 20 mM
Tris/HCl, pH 7.6, 2 mM MgCl2, 1 mM DTT, and 0.2 M KCl. The column was calibrated with the
following molecular mass markers; blue dextran, The Primary Structure of the TK2 Gene Clarified the mt Location of
TK2--
Biochemical and cell fractionation studies have shown that
TK2 is a mitochondrial enzyme (11, 12). However, controversy exists
regarding the existence of a cytosolic form of TK2 (13), and
furthermore, two published human TK2 cDNA sequences do not include
a mt leader sequence (10, 14). Earlier attempts to clone the 5'-end of
human TK2 cDNA were unsuccessful; however, recent methodological
development (15) enabled amplification of the TK2 5'-cDNA sequence,
which has a very high GC content. The mt leader sequence of human TK2
showed high homology with the mouse TK2 sequence (16) but they differ
in the presumed mitochondrial cleavage sites (Fig.
1A). The N-terminal peptide sequence of human TK2 purified from brain (10) showed that amino acid
number 37 was a tyrosine. However, direct translation of the cDNA
sequence and the genomic DNA sequence give an arginine in this
position. At present we can not explain this discrepancy, but one
possibility is that the Arg-37 is post-translationally modified and
eluted in the position of Tyr in the sequencing procedure. It has been
observed earlier with the prion protein that a Tyr and Arg were
inverted in the peptide sequence due to post-translational modification
of the Arg residue (17, 18).
We have partially sequenced the TK2 genomic DNA fragments, mainly the
exons and the intron/exon boundaries, and our results agree with the
published human genome sequence (19). The human TK2 gene occupies a
45-kb DNA fragment on chromosome 16 and consists of 10 exons of 32 bp
up to 1304 bp and 9 introns of 0.572 kb up to 11.1 kb (Fig.
1B). The mt leader sequence is coded by exon 1, and the
entire 3'-untranslated region by exon 10. The 5' sequences of the TK2
isoform A and B reported earlier (14) are part of the intron sequences,
suggesting that these isoforms are alternatively spliced mRNA
products that may not be translated. Therefore, we may conclude that
TK2 fulfils the criteria for being a mitochondrial enzyme.
Preparation of wt and Mutant TK2 Proteins--
A site-directed
mutagenesis method was used to produce the two TK2 mutants, H121N and
I212N, previously numbered as His-90 and Ile-181, which were shown to
be linked to severe mtDNA depletion disorder (8). These mutant enzymes
were expressed in Escherichia coli strain BL21 (DE3) plysS
at high level and purified by one step affinity chromatography to more
than 99% purity by the same procedure as the wild type enzyme (10)
(Fig. 2). There were no differences in
the levels of expression or the yield of purification in case of the wt
and the two mutant enzymes. The N termini of these enzymes contain a
His tag and a thrombin cleavage site, and they were characterized
without removal of the His tag since the presence of the His tag did
not affect the kinetic properties of TK2 in previous studies
(10).
To rule out possible effects of the mutations of the TK2 on overall
structure, purified wt and the two mutant proteins were subjected to
gel filtration chromatography on a Superdex 200 column. Wild type TK2
activity eluted as a broad peak, coinciding with the UV absorbance peak
at a molecular mass range of 30-40 kDa. Both H121N and I212N mutant
TK2 proteins eluted at similar positions during gel filtration
chromatography (Fig. 3). Even though the I212N mutant protein had very low activity, a significant dThd phosphorylating activity (and UV) peak could be detected (Fig. 3). In
no case were there indications of high molecular forms of TK2. We
conclude from this experiment that the mutant enzymes and wt TK2
apparently have similar subunit interaction and are most likely dimers.
Thus, there was no evidence for major structural alterations in TK2 as
a result of the two point mutations. The unusual elution behavior of
TK2 on size exclusion chromatography has been observed earlier (20).
Similarly, mouse TK2 and Drosophila melanogaster
deoxynucleoside kinase (dNK) also showed different molecular sizes
depending on the type of columns used in gel filtration chromatography
(16, 21, 22), and this may be related to the interaction between
TK2-like enzymes and the gel matrix.
The Kinetic Properties of wt and Mutant TK2--
The I212N mutant
showed about 100-fold lower Vmax values with
both dThd and dCyd as substrates as compared with wt TK2 when ATP was
in excess. The Km value for dThd was similar to that
of wt TK2, while the Km value for dCyd was very high
(Table I). Overall this mutant enzyme had
about 1% the efficiency (Vmax/Km value) with dThd and
less than 1% with dCyd as compared with wt TK2 (Table I).
The H121N mutant showed a more complex kinetic pattern, and
surprisingly both the Km and
Vmax values with dThd as substrate were only
somewhat decreased, which resulted in an efficiency similar to wt TK2.
However, with dCyd as substrate the Vmax value was only about 30% of that of wt TK2, while the Km
value was similar (Table I). Another significant change with the H121N mutant is the loss of negative cooperativity with dThd as substrate (Hill constant n = 1).
TK2 has long been shown to have different kinetics toward its two
natural substrates, dThd and dCyd. The phosphorylation of dThd did not
follow Michaelis-Menten kinetics but showed negative cooperativity as
indicated by biphasic Eadie-Hofstee plot (v versus v/s) and
a Hill constant n < 1. In contrast, the
phosphorylation of dCyd followed Michaelis-Menten kinetics and the
Eadie-Hofstee plot for dCyd phosphorylation was a straight line, and
the Hill constant n = 1 (11, 21). To compare the
efficiency of the wt and mutant TK2 enzymes, the Km
value (13 µM) for wt TK2 was calculated by using the
Michaelis-Menten equation; therefore it was apparent, while the
Km value (5.7 µM) for dThd in the case
of the H121N mutant was the true value (Table I). We conclude that the
I212N mutant almost completely lacks enzyme activity, while the H121N
mutant TK2 shows normal activity with dThd, 70% reduced activity with
dCyd as substrate, and an altered cooperativity with dThd as substrate.
These mutant enzymes were also characterized with regard to their
phosphate donor properties with ATP. The Km value for wt TK2 was 25 µM when dThd was the phosphate
acceptor, which is similar to the Km value (38 µM) reported earlier for native TK2 (24). The
Km values for ATP of the H121N mutant with
saturating dThd or dCyd were 3 to 4-fold higher than that of wt TK2,
while the Vmax values were similar with dThd and reduced by 60% with dCyd (Table II). The
I212N mutant showed very low Vmax values and 2 to 4-fold increased Km values for ATP with dThd and
dCyd as compared with those of wt TK2 (Table II). The conclusions from
these experiments are that the I212N mutant showed very low catalytic
rates with all substrates, while the H121N mutant showed a 10-fold
reduced capacity to use dCyd as phosphate acceptor when ATP was a
limiting phosphate donor.
To further clarify the kinetic properties of the H121N mutant a series
of substrate and feedback inhibitor studies were performed (Table
III). The most clear cut difference
between the mutant and wt TK2 was the competition between dThd and dCyd
as alternative substrates. Since the dThd phosphorylation by wt TK2 was
negatively cooperative, double reciprocal plots (1/V versus
1/S) in the presence/absence of dCyd were not linear, which made it
difficult to interpret the mode of inhibition by dCyd. To simplify this
we chose the data sets from the low concentration range where the
double reciprocal plots were linear to analyze the mode of inhibition.
Plots of the intercepts and slopes versus dCyd concentration
were linear, which indicated that the inhibition by dCyd was
non-competitive, and the Ki value was 40 µM (Fig. 4 and Table III).
For the H121N mutant enzyme double reciprocal plots of dThd
phosphorylation over the entire concentration range were linear. The
slope of intercepts versus dCyd concentration plot was zero,
which suggested that dCyd competitively inhibited dThd phosphorylation,
and the Ki value (4 µM) was 10-fold
lower as compared with that for wt TK2 (Fig. 4 and Table III). The dCyd
phosphorylation by both wt and H121N mutant was competitively inhibited
by dThd at similar concentrations; the apparent Ki
values were 4.9 and 3.6 µM, respectively (Fig. 4 and
Table III).
The feedback inhibitors, dTTP and dCTP, showed similar inhibition
patterns with both wt and the H121N mutant, but the
Ki values, with dThd as the substrate, were 2-fold
higher with wt TK2 as compared with the H121N mutant (Table III).
The results with wt TK2 were similar to what was described
earlier with native TK2; dTTP was a competitive inhibitor toward both
dThd and ATP, and the Ki values were in the same
range (3 to 7 µM) (20, 23-25). TK2 as well as all other
deoxynucleoside kinases are feedback-inhibited by their end products,
i.e. deoxynucleoside triphosphates. These feedback
inhibitors probably act as bisubstrate analogous and not as classical
allosteric inhibitors binding to separate effector site on the enzyme
(1). Earlier studies with native TK2 and our kinetic data with
recombinant enzymes indicated that the deoxynucleoside triphosphates
could bind to the phosphate acceptor site since they showed competitive
inhibition with the phosphate acceptor. This possibility has been
proven by the recent three-dimensional structure determinations for dGK
and D. melanogaster dNK, which showed that ATP as
well as dTTP bound to the phosphate acceptor site of the active enzymes
(3, 9).
These results showed again relatively subtle alterations in the kinetic
properties of the H121N mutant, and it was manifested as an apparently
increased capacity of dCyd to compete with dThd as substrate. In case
of wt TK2, dCyd is a relatively inefficient competitor of dThd
phosphorylation. This result is similar to what was reported earlier
with purified lymphoblast TK2 (20), but in that study the
Ki value (630 µM) for dCyd was about
10-fold higher than in the present one using wt TK2. We do not know the
reason for the difference between the recombinant TK2 used here and the
purified lymphoblast enzyme, but it is most likely related to the
differences in enzyme preparations. Overall this phenomenon is probably
related to the negative cooperativity observed with dThd as substrate
for TK2 (1, 2, 20). The mutant enzyme behaved more according to what
could be expected by an alternate substrate in a classic enzyme
competition experiment.
A major goal in this study was to clarify the molecular background for
the mitochondrial location of TK2 by identifying the mt translocation
signal in the TK2 gene sequence. Furthermore, we wanted to mimic the
situation in patients with point mutations in TK2 who suffered from
severe myopathy due to the mtDNA depletion disorder by in
vitro mutagenesis and kinetic characterization of pure recombinant
TK2 preparations. The kinetic results with the recombinant enzymes
strongly support the conclusions made in the earlier study with
mitochondrial extracts from the mtDNA depletion patients (8).
The relative rates of dThd and dCyd phosphorylation carried out by TK2
in the physiological situation as well as the action of feedback
inhibition of deoxynucleoside triphosphates most likely regulate the
size of dTTP and dCTP pools in mitochondria as illustrated in Fig.
5. The I212N mutant enzyme had severely
decreased activity, while the H121N mutant had a reduced capacity to
use dCyd as substrate, and these changes are apparently due to
inefficient ATP/Mg2+ binding and catalysis. These results
correlated well with the clinical severity of the pathologic effect of
specific TK2 mutations: the patients with I212N mutation had severe
myopathic changes of muscle histology, while the patients with H121N
mutation had relatively mild symptom and later onset of myopathy (8,
26). Recently two novel TK2 mutations, T77M and I22M, were identified in patients with mtDNA depletion syndrome and the pathogenicity of
these mutations was confirmed by reduced TK2 activity in muscle extracts (27).
Native TK2 had been shown to have negatively cooperative kinetics with
ATP as substrate, and the Km value for ATP was
dependent on the concentration of dThd; at physiologically relevant
concentration of dThd (5 µM) the Km
value for ATP was 200 µM (25). Although it has not been
possible to measure the ATP content in the mitochondria of these
patients at the time of biopsy, oligomycin-sensitive ATP synthase
(complex V) activity was measured (the direction of ATP hydrolysis) and
found to be severely decreased in all patients (web supplement Table A
in Ref. 8). Additionally, oxygen consumption of mitochondria from the
patient carrying the His mutation was measured in freshly isolated
muscle mitochondria. The oxygen consumption was severely decreased with
all substrates tested, which imply that the respiratory chain was
malfunctioning also in vivo and that the formation of a
proton gradient across the mitochondrial inner membrane was hampered.
It is therefore reasonable to assume that this will lead to a low
mitochondrial ATP content (web supplement Table A in Ref. 8), which in
turn may lead to an even lower efficiency of the mutant TK2 enzymes
than what was observed in vitro with the recombinant
enzymes. Therefore, the I121N mutation probably resulted in very low mt
dCTP and dTTP pools, while the H212N mutation most likely leads to
normal dTTP pool but very low dCTP pool as compared with the dATP and
dGTP pools (Fig. 5). The inability to salvage deoxynucleosides and the
imbalance in mt dNTP pools should preferentially affect tissues/or
cells where no uptake of dNTPs from the cytosol is possible due to the
lack of de novo DNA precursor synthesis (1, 2). Earlier
study has suggested that mtDNA copy number may be regulated by tightly
controlled mitochondrial dNTP pools (26). The altered mt dNTP pools can be expected to give increased levels of mutations and depletions of
mtDNA; the latter has been observed in the patients with myopathy (8,
27-29). The onset of the mtDNA depletion syndrome caused by TK2
mutation is probably dependent on mtDNA turnover and other metabolic
situations in the tissues, e.g. the developmental
down-regulation of key enzymes such as ribonucleotide reductase. One of
the future tasks is to explain why deficiency in TK2 leads to
functional defects of muscles. The establishment of a relevant animal
model system would be an important step toward this goal and may in the
future also enable development of gene and chemotherapies for this
type of devastating mt disorders.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-galactopyranoside at
37 °C. Wild type TK2 expression was performed in parallel as a
control. The bacteria were harvested by centrifugation at 3000 × g for 15 min at 4 °C, and the pellet was resuspended in
lysis buffer (50 mM Tris/HCl, pH 7.6, 0.5 M
NaCl, 0.5% Triton X-100, and 140 µM phenylmethylsulfonyl
fluoride). Total proteins were extracted by freezing and thawing three
times and followed by sonication at 16 A for 30 s. The lysate was
centrifuged at 100,000 × g for 90 min at 4 °C. Then
the enzymes were purified by metal affinity column chromatography
(TALON, Clontech) essentially as described in the
manufacturer's instruction. Both wild type and mutant enzymes were
eluted with 0.25 M imidazole in buffer containing 50 mM Tris/HCl, pH 7.9, 0.5 M NaCl, 0.1% Triton
X-100, and 10% glycerol. The fractions containing TK2 activity were
pooled and concentrated by using a Centriprep device (Millipore).
Aliquots of purified enzymes were analyzed by 12% SDS-PAGE, and
protein concentration was determined by Bio-Rad protein assay using BSA as standard.
-amylase (200 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa), and cytochrome
c (12.4 kDa) (Sigma), and the active molecular masses of the
wt TK2 and the mutant proteins were estimated from the standard curve.
Fractions were collected and assayed for dThd kinase activity.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (14K):
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Fig. 1.
A, N-terminal amino acid sequence
alignment of human and mouse TK2. Arrows indicate the
mitochondrial cleavage sites and the underlined sequence is
the N-terminal sequence of processed mitochondrial TK2 (11, 17). The
letter Y above the human TK2 sequence indicates the amino acid found in
purified human TK2 (11); B, the human TK2 gene
structure.
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[in a new window]
Fig. 2.
SDS-PAGE of purified wt and H121N and I212N
mutant TK2 enzymes. Lanes 1 and 6, molecular mass
markers; lane 2, uninduced culture; lanes 3,
4, and 5, induced culture of wt, H121N, and I212N
respectively; lanes 7, 8, and 9,
purified wt, H121N, and I212N enzymes respectively.
View larger version (19K):
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Fig. 3.
Gel filtration chromatography on Superdex 200 column. The dThd activity with wt (circles), H121N
mutant (squares), and I212N mutant (triangles)
TK2 enzymes. The elution positions of -amylase (200 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa) are indicated with arrows. Vo, void
volume.
Kinetic parameters of human wild type TK2 and the H121N and I212N
mutant enzymes
The kinetic parameters of wild type and mutant TK2 enzymes with
ATP/MgCl2
The inhibition pattern and Ki values of wild type and the H121N
mutant TK2 with substrates and end products
View larger version (21K):
[in a new window]
Fig. 4.
Double reciprocal plots (1/V
versus 1/S) of competition studies between dThd and
dCyd. Upper panel, dThd phosphorylation by wt TK2
(left) and H121N mutant enzyme (right) in the
presence of dCyd; insets are the plots of intercepts
(circles) and slopes (triangles)
versus dCyd concentration (µM); lower
panel, dCyd phosphorylation by wt TK2 (left) and H121N
mutant enzyme (right) in the presence of dThd. The
concentrations of inhibitors are indicated by numbers beside the lines
(µM).
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Fig. 5.
Schematic illustration of the interaction
among the substrates and feedback inhibitors for the role of TK2 in the
regulation of the DNA precursor pools in mitochondria.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Medical Research Council (to S. E. and L. W.), the Swedish Natural Science Research Council (to S. E.), the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (to L. W. and S. E.), and the Israel Science Foundation, administered by the Israeli Academy of Sciences and Humanities (to A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 46-18-471-41-19; Fax: 46-18-55-07-62; E-mail: liya.wang@vmk.slu.se.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M206143200
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ABBREVIATIONS |
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The abbreviations used are: mt, mitochondrial; dNTP, deoxynucleoside triphosphates; TK, thymidine kinase; dCK, deoxycytidine kinase; dGK, deoxyguanosine kinase; dThd, thymidine; dCyd, deoxycytidine; dAdo, deoxyadenosine; dGuo, deoxyguanosine; dNK, deoxynucleoside kinase; BSA, bovine serum albumin; DTT, dithiothreitol.
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REFERENCES |
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