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Thymidine kinase

Thymidine kinase (TK) is a family of phosphotransferase enzymes that catalyze the transfer of a phosphate group from ATP to thymidine, forming thymidine monophosphate (dTMP), a critical precursor for deoxythymidine triphosphate (dTTP) synthesis in the pyrimidine salvage pathway.^[1] This reaction is essential for maintaining deoxyribonucleotide pools required for DNA replication, repair, and cell proliferation.^[2] In humans, two primary isozymes exist: the cytosolic TK1, which is highly regulated and active primarily during the S phase of the cell cycle, and the mitochondrial TK2, which supports dNTP homeostasis in quiescent cells and during mitochondrial DNA synthesis.^[3]^[4] Human TK1, encoded by the TK1 gene on chromosome 17q25.3, functions as a homotetramer with a subunit molecular weight of approximately 25 kDa, featuring a bilobal structure with an N-terminal domain for ATP binding and a C-terminal domain for substrate recognition, including key residues like Glu98, Phe128, and Tyr181 in the active site.^[1] Its expression and activity are tightly controlled by cell cycle checkpoints, including E2F transcription factors for upregulation in G1/S transition and ubiquitin-mediated degradation via the anaphase-promoting complex during G2/M phase, ensuring activity aligns with DNA synthesis demands.^[3] TK2, encoded by TK2 on chromosome 16q21, is a monomer-dimer equilibrium enzyme with broader substrate specificity, phosphorylating not only thymidine but also deoxycytidine and deoxyuridine, and it remains constitutively expressed to sustain mitochondrial function.^[4] Beyond mammalian forms, thymidine kinases are found in viruses such as herpes simplex virus (HSV), where viral TK exhibits promiscuous substrate specificity that activates nucleoside analogs like acyclovir for antiviral therapy.^[5] TK1 levels in serum or tissues serve as a proliferation marker, elevated in malignancies like breast, lung, and lymphoma cancers, enabling its use in diagnosis, prognosis, and monitoring treatment response through assays such as the TK-Liaison or DiviTum platforms.^[3] Mutations in TK2 cause mitochondrial DNA depletion syndromes, leading to severe neuromuscular disorders, with therapeutic strategies including deoxynucleoside supplementation; no known human TK1 deficiency syndromes have been reported, highlighting TK1's essential role in development.^[6]^[3] Overall, thymidine kinases underscore the balance between de novo and salvage nucleotide pathways, with implications spanning basic cell biology to clinical oncology and virology.

Discovery and History

Initial Discovery

Thymidine kinase was first identified in the 1950s through studies on the incorporation of thymidine into DNA in mammalian cells, particularly in proliferating tissues. Maurice Friedkin, a pioneer in nucleotide metabolism, along with Donald Tilson and DeWayne Roberts, conducted seminal experiments using radiolabeled [14C]-thymidine to trace its uptake and conversion in embryonic tissues, bone marrow, and tumor cells, demonstrating that thymidine undergoes initial phosphorylation to thymidine monophosphate as a prerequisite for DNA synthesis.^[7] These findings highlighted the enzyme's role in salvage pathways, with higher activity observed in rapidly dividing cells such as those from rabbit bone marrow and Ehrlich ascites tumors.^[7] Building on this, researchers in the late 1950s further characterized the phosphorylation activity. In 1959, F.J. Bollum and V.R. Potter isolated soluble enzymes from regenerating rat liver extracts that converted thymidine to thymidine phosphates and subsequently incorporated them into DNA, providing direct evidence of kinase-mediated activity in mammalian systems.^[8] Shortly thereafter, S.M. Weissman, R.M.S. Smellie, and J. Paul demonstrated thymidine phosphorylation in cell-free extracts from mammalian tissues, confirming the enzyme's presence and specificity using ATP as the phosphate donor.^[9] Similar observations were reported in proliferative states, reinforcing the link between kinase activity and DNA biosynthesis. The first purification and partial characterization of thymidine kinase occurred in the early 1960s, establishing it as a distinct enzyme classified under EC 2.7.1.21. Efforts focused on extracts from fetal and tumor tissues, where activity was highest, yielding partially purified fractions that exhibited specific phosphorylation of thymidine without significant contamination by other kinases.^[10] These advancements, including studies on enzyme kinetics and tissue distribution, solidified thymidine kinase's identity as a key regulator in nucleotide metabolism.

Key Milestones and Classification Development

In the 1960s, thymidine kinases from viruses such as herpes simplex virus (HSV) were identified in infected cells, with initial descriptions by Dubbs and Kit demonstrating distinct enzymatic activity.^[11] This laid the foundation for later recognition of their broader substrate specificity, including phosphorylation of nucleoside analogs like acyclovir, which was characterized in the 1970s and enabled antiviral therapies by the early 1980s. In the 1970s, cell fractionation studies revealed the existence of distinct cytosolic and mitochondrial forms of thymidine kinase, marking a pivotal advancement in understanding their compartmentalization. Researchers demonstrated that mammalian mitochondria possess a genetically distinct thymidine kinase activity capable of exclusively labeling mitochondrial DNA, independent of the cytosolic enzyme, through subcellular isolation techniques in mouse L cells and other tissues.^[12] This distinction was further supported by observations of thymidine kinase persistence in mitochondrial fractions of kinase-deficient cell lines, confirming the mitochondrial form's autonomy. Concurrently, studies identified the cytosolic form's regulation, peaking during the S-phase of the cell cycle, which highlighted its role in nuclear DNA synthesis. The 1980s brought significant progress through molecular cloning, beginning with the human cytosolic thymidine kinase 1 (TK1) gene, sequenced in 1983, which enabled detailed analysis of its promoter and S-phase-specific expression.^[13] This cloning revealed TK1's tight cell cycle regulation, contrasting with the constitutive expression of the mitochondrial form. Cloning of the mitochondrial thymidine kinase 2 (TK2) gene occurred later, in 1997, allowing chromosomal localization to 16q22 and insights into its broader substrate range for deoxyribonucleosides like deoxycytidine and deoxyuridine.^[14] These efforts underscored kinetic differences, with TK1 showing higher affinity for thymidine and ATP dependence tied to proliferation, while TK2 exhibited stable, non-proliferative activity. Post-2000 developments included crystal structures of human TK1 (e.g., PDB 1XBT in 2004 and 1W4R in 2005), providing mechanistic insights into its tetrameric architecture and substrate binding.^[15] For TK2, structural modeling in the 2010s and genetic studies on mutations linked to mitochondrial disorders in the 2020s further refined understanding of its role in mtDNA maintenance. Nomenclature evolved from a singular "thymidine kinase" designation to the standardized TK1/TK2 distinction, based on subcellular localization (cytosolic vs. mitochondrial), kinetic properties, and regulatory patterns, as formalized in comprehensive reviews.^[16] This classification facilitated targeted research into isozyme-specific functions and therapeutic applications.

Classification and Isozymes

Cytosolic Thymidine Kinase 1 (TK1)

Cytosolic thymidine kinase 1 (TK1) is encoded by the TK1 gene located on the long arm of human chromosome 17 at position 17q25.3. The gene consists of seven exons spanning approximately 13 kb of genomic DNA.^[17] The encoded protein is a 234-amino-acid polypeptide with a molecular weight of approximately 25 kDa per subunit, forming a homotetrameric structure essential for its enzymatic activity; this quaternary assembly, totaling around 100 kDa, features an α/β-domain resembling ATPase motifs in the RecA structural family.^[18] In quiescent cells, TK1 predominantly exists as inactive dimers, transitioning to the active tetrameric form upon cell cycle progression.^[19] TK1 expression is tightly linked to cell proliferation, exhibiting S-phase specificity where it peaks during active DNA replication to support deoxythymidine monophosphate (dTMP) production via the salvage pathway. In quiescent (G0) cells, TK1 levels are negligible or absent, reflecting its role as a proliferation marker rather than a constitutive enzyme.^[20] Elevated TK1 expression is a hallmark of rapidly dividing cells, including those in various tumors such as lung, breast, and prostate cancers, where it correlates with increased DNA synthesis rates and poor prognosis.^[19] Regulation of TK1 occurs at multiple levels to align its activity with the cell cycle. Transcriptionally, the TK1 promoter contains E2F-binding sites that drive expression upon retinoblastoma protein (pRB) hyperphosphorylation, releasing E2F transcription factors to activate S-phase genes during G1/S transition. Post-translationally, TK1 stability is controlled by ubiquitin-proteasome-mediated degradation at the G2/M phase, facilitated by a C-terminal KEN box motif that serves as a recognition signal for the anaphase-promoting complex/cyclosome (APC/C), ensuring rapid enzyme clearance after DNA synthesis. This degradation prevents excess dTTP accumulation and prepares cells for mitosis.^[19] Kinetic properties of TK1 underscore its specificity for pyrimidine deoxyribonucleosides. The enzyme exhibits a Michaelis constant (K_m) for thymidine in the range of 0.5–5 μM, with optimal activity at physiological ATP concentrations (mM range), enabling efficient salvage of exogenous thymidine. TK1 shows high substrate specificity for deoxythymidine and deoxyuridine, with limited affinity for purine deoxyribonucleosides like deoxyguanosine or deoxyadenosine, distinguishing it from broader-spectrum deoxyribonucleoside kinases. TK1 demonstrates evolutionary conservation across eukaryotes, with orthologs identified in mammals, yeast, and other unicellular eukaryotes, reflecting its fundamental role in DNA precursor salvage. While bacterial thymidine kinases (e.g., tdk in Escherichia coli) share structural similarities such as multimeric assembly and catalytic domains, they lack direct sequence homology to eukaryotic TK1, suggesting divergent evolution within prokaryotes.

Mitochondrial Thymidine Kinase 2 (TK2)

Mitochondrial thymidine kinase 2 (TK2), encoded by the nuclear TK2 gene located on human chromosome 16q21, is a deoxyribonucleoside kinase essential for maintaining mitochondrial nucleotide pools. The protein consists of monomers approximately 30 kDa in size that assemble into a homodimeric structure, with the active enzyme localized to the mitochondrial matrix where it facilitates the salvage phosphorylation of pyrimidine deoxynucleosides.^[21] TK2 exhibits constitutive expression across all tissues, remaining independent of the cell cycle and providing a stable supply of deoxynucleotide precursors for ongoing mitochondrial DNA (mtDNA) synthesis. This housekeeping role ensures continuous mtDNA maintenance in post-mitotic and non-proliferating cells, contrasting with the cell cycle-dependent expression of cytosolic TK1. High levels of TK2 transcripts are particularly noted in metabolically active tissues such as testis and muscle, underscoring its broad physiological necessity.^[22] Regulation of TK2 activity occurs through allosteric mechanisms involving ATP as a phosphate donor and feedback inhibition by deoxyribonucleoside triphosphates (dNTPs) like dTTP and dCTP, which prevent excessive accumulation of mitochondrial dNTP pools. Compared to TK1, TK2 demonstrates broader substrate specificity, efficiently phosphorylating not only thymidine but also deoxycytidine and deoxyuridine to their respective monophosphates. Its kinetic properties include a relatively high Michaelis constant (Km) for thymidine of approximately 15–50 μM, reflecting lower affinity than TK1 and adaptation to steady-state substrate levels in mitochondria, alongside inhibition by dTTP to fine-tune precursor availability.^[23]^[24] Mutations in the TK2 gene, with over 50 distinct variants identified to date, disrupt enzyme function and lead to impaired mtDNA maintenance, resulting in mitochondrial disorders such as TK2 deficiency (detailed in the Genetic Deficiency and Disorders section). These mutations often affect catalytic residues or dimer stability, highlighting TK2's critical role in mitochondrial homeostasis.^[25]^[26]

Viral and Other Thymidine Kinases

Viral thymidine kinases, particularly those from herpesviruses, play critical roles in nucleotide salvage during infection by phosphorylating a wide range of substrates to support viral DNA replication. The thymidine kinase encoded by herpes simplex virus type 1 (HSV-1 TK) is a homodimeric enzyme with a broad substrate specificity that extends beyond thymidine to include antiviral nucleoside analogs like acyclovir, enabling selective drug activation in infected cells.^[27]^[28] The HSV-1 TK gene, located on the viral genome, is essential for efficient viral replication and establishment of latency in sensory neurons.^[29]^[30] Related enzymes in other herpesviruses exhibit similar but distinct properties. Varicella-zoster virus thymidine kinase (VZV-TK) shares structural homology with HSV-1 TK, forming a homodimer capable of phosphorylating thymidine and analogs such as (E)-5-(2-bromovinyl)-2′-deoxyuridine, though with varying kinetic efficiencies that influence antiviral susceptibility.^[31]^[32] In contrast, Epstein-Barr virus thymidine kinase (EBV-TK) demonstrates thymidylate kinase activity but lacks phosphorylation capability for acyclovir or ganciclovir, distinguishing it from alphaherpesvirus TKs and limiting its utility in certain therapeutic contexts.^[33]^[34] Bacterial and parasitic thymidine kinases differ markedly in specificity and structure from their viral counterparts. In Escherichia coli, the thymidine kinase encoded by the tdk gene is highly specific for thymidine and deoxyuridine, facilitating deoxyribonucleotide salvage without the broad analog acceptance seen in viral enzymes.^[35]^[36] The thymidine kinase from the parasite Giardia intestinalis, characterized in a 2022 study, belongs to a divergent TK1 family subclass with a unique bifunctional structure of two weakly homologous domains, displaying high affinity for thymidine (K_m ≈ 0.5 μM) and proving essential for parasite DNA synthesis, thus positioning it as a selective drug target.^[37] Compared to mammalian thymidine kinases, viral variants are notably more promiscuous toward nucleoside analogs, a property exploited in antiviral therapies, while often exhibiting lower thermal stability that affects their conformational dynamics in host environments.^[38]^[39] Evolutionarily, horizontal gene transfer has contributed to the acquisition of thymidine kinase genes in certain pathogens; for instance, in the apicomplexan parasite Cryptosporidium parvum, the TK gene traces to a proteobacterial origin, enhancing pyrimidine salvage pathways.^[40] Similar transfers occur in microsporidian fungi, underscoring HGT's role in pathogen adaptation.^[41]

Biochemical Function

Catalytic Mechanism

Thymidine kinase (TK), classified under EC 2.7.1.21, catalyzes the phosphorylation of thymidine to deoxythymidine monophosphate (dTMP) using ATP as the phosphate donor, yielding the reaction thymidine + ATP ⇌ dTMP + ADP in the presence of Mg²⁺ as a cofactor.^[10] The enzyme follows an ordered sequential bi-bi kinetic mechanism, in which thymidine binds first to the active site, followed by ATP, with ADP released prior to dTMP, distinguishing it from ping-pong mechanisms observed in some other kinases.^[42] This sequential binding ensures efficient substrate positioning for phosphoryl transfer, with Michaelis constants reported as approximately 5 μM for thymidine and 90 μM for ATP in human liver TK.^[42] The active site of human cytosolic TK1 (hTK1) is located in a cleft between an α/β-domain and a lasso-like loop stabilized by a structural zinc ion, as revealed by the crystal structure (PDB: 1XBT) determined at 2.4 Å resolution in complex with the feedback inhibitor dTTP.^[4] Thymidine binds through hydrogen bonds from its ribose 3'- and 5'-hydroxyls and base (O2 and N3 atoms) to main-chain atoms in the lasso loop, with the thymine base stacking against conserved arginine and tyrosine residues and its methyl group accommodated in a hydrophobic pocket formed by methionine, leucine, and other side chains.^[4] ATP coordination involves conserved aspartate residues, such as those in the P-loop motif of the α/β-domain, which help position the Mg²⁺ ion to bridge the β- and γ-phosphates of ATP, facilitating substrate alignment.^[43] In the catalytic mechanism, Glu-98 acts as a general base to deprotonate the 5'-hydroxyl group of thymidine, enhancing its nucleophilicity for direct attack on the γ-phosphate of Mg-ATP, resulting in phosphoryl transfer and formation of dTMP while releasing ADP.^[4] A cluster of arginine and lysine residues stabilizes the transition state by interacting with the transferring phosphate, and the structural zinc maintains the lasso loop in a closed conformation over the active site during catalysis.^[44] This process is tightly regulated by feedback inhibition from dTTP, which binds competitively to the thymidine site with a Ki of approximately 13.5 μM, preventing excessive dTMP accumulation.^[42] Nucleoside analogs such as azidothymidine (AZT) act as competitive inhibitors or alternative substrates by binding to the thymidine site, with hTK1 phosphorylating AZT to AZT-monophosphate at about 50% the efficiency of thymidine.31547-3/pdf)

Role in Nucleotide Salvage Pathway

Thymidine kinase catalyzes the phosphorylation of thymidine to deoxythymidine monophosphate (dTMP) in the nucleotide salvage pathway, enabling cells to recycle exogenous or endogenously generated thymidine nucleosides into usable deoxyribonucleotide precursors. This salvage route contrasts with the de novo synthesis pathway, which generates dTMP from uridine derivatives through ribonucleotide reductase (RNR)-mediated reduction of UDP to dUDP, followed by conversion to dUMP and methylation by thymidylate synthase. By directly utilizing thymidine, the salvage pathway initiated by thymidine kinase bypasses the RNR-dependent step, providing an energetically efficient alternative for dTTP production essential for DNA replication.^[45]^[19] Following dTMP formation, the salvage pathway integrates with downstream enzymes: thymidylate kinase phosphorylates dTMP to dTDP, and nucleoside diphosphate kinase further converts dTDP to dTTP, replenishing the deoxyribonucleotide triphosphate (dNTP) pool. This dTTP exerts allosteric feedback inhibition on RNR, modulating de novo synthesis to prevent dNTP imbalances and maintain cellular nucleotide homeostasis. Thymidine kinase activity itself is also regulated by dTTP binding, which inhibits further phosphorylation and fine-tunes salvage flux.^[45]^[19] In cellular metabolism, the salvage pathway is particularly vital in proliferating cells, where cytosolic thymidine kinase levels are elevated to efficiently supply dTTP during active DNA synthesis, while a mitochondrial branch supports dNTP needs for mitochondrial DNA maintenance. This mechanism contributes a significant portion of the dTTP pool in mammalian cells, with reliance varying by tissue and physiological state. Dysregulation of the salvage pathway can lead to dNTP pool imbalances, resulting in replication stress, increased DNA damage, and elevated mutagenesis rates during genome duplication.^[45]^[46]^[19]

Physiological Roles

In Cell Proliferation and DNA Synthesis

Thymidine kinase 1 (TK1) is predominantly expressed and upregulated during the S phase of the cell cycle, where it plays a critical role in facilitating DNA replication by phosphorylating thymidine to thymidine monophosphate (dTMP), which is subsequently converted to deoxythymidine triphosphate (dTTP). This upregulation ensures an adequate supply of dTTP, a essential deoxyribonucleotide triphosphate (dNTP) substrate for replicative DNA polymerases such as polymerase δ (Pol δ) and polymerase ε (Pol ε), which synthesize the leading and lagging strands during DNA replication forks.^[3]^[47] TK1 activity is tightly regulated to match the demands of rapid DNA synthesis, with its levels peaking to support the high dTTP consumption rates in proliferating cells.^[48] TK1 also influences cell cycle checkpoints by modulating replication dynamics; balanced dTTP pools maintained by TK1 help regulate replication fork progression, preventing excessive slowing or stalling that could trigger DNA damage responses. Depletion of TK1 leads to dNTP imbalances, which slow replication fork speeds and induce S-phase arrest, as cells activate checkpoints like the ATR-Chk1 pathway to halt progression until nucleotide availability is restored.^[49]^[50] In contrast, in non-dividing or quiescent cells, TK2 provides sufficient dTMP for low-level DNA maintenance and repair synthesis, such as in mitochondrial DNA or nuclear base excision repair, without the need for cell cycle-dependent upregulation.^[51]^[52] Experimental studies underscore TK1's necessity for proliferation; TK1 knockout in hepatocellular carcinoma cell lines, such as Hep3B, significantly reduces cell growth and viability due to impaired dTTP synthesis and replication defects.^[53] Similarly, analogs like bromodeoxyuridine (BrdU), used to label newly synthesized DNA, require TK1 for phosphorylation and incorporation into replicating DNA, as cells lacking TK1 fail to integrate BrdU efficiently.^[54] Quantitatively, TK1 expression levels strongly correlate with the growth fraction of cell populations, with proliferating tumor cells often exhibiting 10- to 20-fold higher TK1 activity compared to normal tissues, reflecting their elevated DNA synthesis demands.^[55]^[16]

During Development and Tissue Expression

Thymidine kinase 1 (TK1) exhibits dynamic expression patterns during embryonic development, reflecting its association with cell proliferation. In mouse embryos, TK1 is expressed in early conceptus stages, including ectoderm and mesenchyme, and persists in proliferating structures such as the peripheral nervous system (derived from neural crest) and limb buds. At embryonic day 14.5, RNA in situ hybridization reveals weak expression in cranial ganglia, moderate levels in forelimb and hindlimb buds, and strong signals in the spinal cord and midbrain, underscoring its role in rapidly dividing embryonic tissues. In contrast, mitochondrial thymidine kinase 2 (TK2) maintains relatively constant expression throughout embryogenesis to support steady-state mitochondrial DNA (mtDNA) replication, with ubiquitous distribution ensuring nucleotide availability for non-proliferative mtDNA maintenance.^[56]^[57]^[21] Postnatally, TK1 expression declines markedly in quiescent tissues, aligning with reduced cell division rates. In rat models, TK1 activity in liver tissue peaks during fetal stages and becomes undetectable or significantly reduced post-neonatally, while TK2 persists at detectable levels. This pattern extends to other organs: TK1 levels decrease in the brain and heart, where cells are largely post-mitotic, but remain elevated in regenerative tissues such as liver and testis, which retain proliferative capacity. TK1 is particularly enriched in hematopoietic progenitors within bone marrow and thymus, supporting rapid turnover in blood cell lineages. TK2, however, shows stable, ubiquitous expression across postnatal tissues, with higher levels in skeletal muscle, liver, pancreas, and brain, where it sustains mtDNA integrity amid varying metabolic demands. In reproductive tissues, estrogen modulates TK1 expression transcriptionally, as observed in breast-derived cell lines, influencing proliferation in hormone-responsive contexts.^[16]^[58]^[59] During aging, TK1 activity exhibits a gradual decline, correlating with overall reduced cellular proliferation. Serum TK1 levels decrease significantly from young adulthood (18–35 years) through middle age (36–60 years) to elderly stages (60–86 years), reflecting diminished regenerative potential in aging tissues. TK2 expression remains stable, playing a critical role in preserving mtDNA integrity, as disruptions in TK2 function lead to progressive mtDNA depletion that mirrors age-related mitochondrial decline. This stability of TK2 underscores its essential, non-proliferative function in long-term cellular maintenance across lifespan stages.^[60]^[61]

Species Distribution and Evolution

Thymidine kinase (TK) enzymes exhibit a broad distribution across kingdoms of life, underscoring their conserved role in nucleotide salvage. In eukaryotes, TK homologs are present in diverse taxa, including plants and animals, where they facilitate the phosphorylation of thymidine to support DNA synthesis. For instance, Arabidopsis thaliana possesses two TK isoforms: cytosolic AtTK1 and mitochondrial AtTK2, both essential for growth, development, and genotoxic stress tolerance.^[62] Similarly, in animals, TK1 (cytosolic) and TK2 (mitochondrial) are widely expressed across vertebrates, with additional lineage-specific expansions in the TK2-like deoxyribonucleoside kinase (dNK) family observed in fish, amphibians, and birds, such as duplicated deoxycytidine kinase (dCK) paralogs.^[63] In fungi, while Saccharomyces cerevisiae lacks a native TK homolog and relies on alternative pathways like thymidylate kinase for nucleotide metabolism, other yeasts such as Schizosaccharomyces pombe also lack native TK and require exogenous expression for thymidine utilization.^[64]^[65] In prokaryotes, TKs are nearly ubiquitous, with phylogenetic diversity reflecting two major groups: those from Gram-positive bacteria closely related to eukaryotic TK1, and a distinct cluster in Gram-negative species like Escherichia coli, where the tdk gene enables efficient thymidine salvage, particularly in pathogenic contexts.^[66]^[67] However, TK is absent in organisms with highly reduced genomes, such as certain Mycoplasma species, which depend on host-derived nucleotides due to their parasitic lifestyle. Viral TKs, exemplified by herpes simplex virus (HSV) TK, are also widespread among DNA viruses like herpesviruses and poxviruses, exhibiting broad substrate specificity adapted for replication in host cells with limited nucleotide pools; these enzymes share evolutionary relatedness with cellular TKs, indicating ancient horizontal gene transfer events.^[68] Evolutionarily, TKs represent an ancient salvage enzyme family, with roots traceable to early prokaryotic lineages and subsequent diversification in eukaryotes. The TK1 family likely originated from bacterial ancestors and is retained in plants and most animals, while the TK2-like dNK family arose from a common progenitor that duplicated prior to the divergence of insects and vertebrates approximately 600 million years ago, leading to specialized enzymes like TK2, dCK, and dGK in metazoans.^[69] In insects such as Drosophila, TK1 was lost, and a single broad-specificity TK2-like dNK suffices, demonstrating retrograde evolution through minimal sequence changes. Parasitic protists like Giardia intestinalis encode a high-affinity TK crucial for DNA synthesis via host nucleotide scavenging, whereas Plasmodium falciparum lacks a native TK and relies on de novo pathways or exogenous supplementation.^[37] Comparative analyses reveal conserved TK1/TK2 architectures in mammals and birds, with fish exhibiting tissue-specific isoform variations that support adaptive nucleotide homeostasis in diverse physiological contexts.^[63]

Genetic Deficiency and Disorders

TK1 Deficiency

TK1 deficiency has not been reported in humans, likely due to the enzyme's essential role in nucleotide salvage during development, potentially resulting in embryonic lethality.^[16] In contrast, TK1 knockout mouse models, which mimic a complete loss of function through targeted gene disruption, provide insight into the consequences of TK1 absence. These models demonstrate autosomal recessive inheritance patterns, as homozygous knockouts exhibit the phenotype while heterozygotes remain unaffected.^[70] In TK1 knockout mice, phenotypes include a compromised immune system with evidence of lymphocyte proliferation defects, shortened lifespan, and progressive kidney failure characterized by glomerular sclerosis. Elevated levels of deoxythymidine in serum reflect impaired salvage phosphorylation, leading to nucleotide pool imbalances that particularly affect rapidly dividing cells such as T lymphocytes, potentially contributing to immune dysfunction. Anemia has not been consistently observed in these models, but replication stress in hematopoietic cells may underlie related proliferative impairments.^[70]^[71] Diagnosis of TK1 deficiency in model systems involves measurement of low enzymatic activity in erythrocytes or tissues, often using radiometric assays to quantify thymidine phosphorylation rates. The TK1 knockout mice were first described in the early 2000s, building on earlier characterizations of the gene's function.^[70] Pathophysiologically, the absence of TK1 disrupts the cytosolic salvage pathway for deoxythymidine monophosphate (dTMP) synthesis, causing deoxythymidine triphosphate (dTTP) depletion and dNTP imbalances that hinder DNA replication and repair, especially in immune cells reliant on high turnover. This leads to increased mutation rates and selective vulnerability in proliferative tissues like the hematopoietic and renal systems.^[71] No specific therapies exist for TK1 deficiency, as human cases are unreported; management in mouse models is supportive, focusing on monitoring renal function and immune status. Nucleoside analogs that depend on TK1 activation, such as those used in antiviral or anticancer treatments, should be avoided to prevent exacerbation of nucleotide imbalances.^[16]

TK2 Deficiency and Mitochondrial Myopathy

TK2 deficiency, also known as thymidine kinase 2-related mitochondrial DNA maintenance defect, is an autosomal recessive disorder caused by biallelic pathogenic variants in the TK2 gene located on chromosome 16q22.1.^[72] Over 150 distinct mutations have been identified worldwide, predominantly missense variants clustered in hotspots such as exons 5 and 8, which encode the substrate-binding and active sites of the enzyme.^[26] Common examples include p.Thr108Met (c.323C>T) and p.Lys202del (c.604_606delAAG), which severely impair enzyme function.^[26] These variants typically reduce TK2 enzymatic activity by 80-100%, with residual activity as low as 3-6% in affected tissues, leading to insufficient phosphorylation of thymidine and deoxycytidine in mitochondria.^[73] Clinically, TK2 deficiency manifests as a phenotypic continuum of mitochondrial myopathy, characterized by progressive skeletal muscle weakness, hypotonia, and respiratory insufficiency.^[72] Onset varies widely: infantile forms (<2 years) present with severe hypotonia and failure to thrive, while juvenile (2-12 years) and late-onset (>12 years) cases often begin with proximal limb-girdle weakness and fatigue, progressing to ventilator dependence.^[26] Additional features may include dysphagia, dysarthria, and sensorineural hearing loss, though cognitive function is generally preserved.^[72] Mitochondrial DNA (mtDNA) depletion, typically to 5-30% of normal levels, predominantly affects skeletal muscle but can involve brain tissue in severe cases, contributing to occasional encephalomyopathic presentations.^[72] Pathophysiologically, diminished TK2 activity depletes the mitochondrial pool of deoxythymidine triphosphate (dTTP) and deoxycytidine triphosphate (dCTP), essential for mtDNA replication and repair.^[25] This imbalance triggers progressive mtDNA loss and multiple deletions, impairing oxidative phosphorylation and energy production in high-demand tissues like muscle.^[26] Histologically, affected muscle fibers exhibit ragged-red appearance due to subsarcolemmal accumulation of dysfunctional mitochondria, alongside cytochrome c oxidase (COX)-deficient fibers.^[72] Diagnosis relies on a combination of clinical evaluation, biochemical assays, and molecular testing. Muscle biopsy often reveals mtDNA depletion, ragged-red fibers, and reduced respiratory chain enzyme activities, particularly complexes I and IV.^[26] Genetic confirmation involves next-generation sequencing of the TK2 gene, identifying biallelic variants.^[72] Elevated levels of deoxythymidine and deoxycytidine in cerebrospinal fluid (CSF) serve as a supportive biomarker, reflecting impaired nucleotide salvage.^[26] Serum creatine kinase is frequently elevated (5-10 times normal), aiding initial suspicion.^[72] Recent therapeutic advances focus on substrate supplementation to bypass TK2 deficiency. In November 2025, the U.S. Food and Drug Administration approved Kygevvi (deoxycytidine and deoxythymidine), the first treatment for TK2 deficiency in adults and pediatric patients with symptom onset at or before 12 years of age. This oral fixed-dose combination therapy, administered at doses up to 400 mg/kg/day, demonstrated in phase 2 trials (e.g., NCT03845712) and compassionate-use studies a 95% reduction in mortality risk, improved survival, enhanced muscle strength, and decreased ventilation requirements, particularly in early-onset cases.^[74]^[25] Preclinical mouse models of Tk2 deficiency show partial restoration of mtDNA levels and 2- to 3-fold lifespan extension with deoxycytidine/deoxythymidine treatment, often combined with adeno-associated virus (AAV9) gene therapy for enhanced efficacy. These interventions represent disease-modifying strategies, though long-term outcomes continue to be evaluated as of late 2025.^[25]

Applications in Medicine and Research

Diagnostic Applications

Thymidine kinase 1 (TK1) functions as a key serum biomarker for assessing cell proliferation in oncology, with elevated levels indicating active tumor growth. In hematological malignancies such as non-Hodgkin lymphoma, serum TK1 activity exceeding 16.6 U/L predicts progression from indolent to aggressive disease and correlates with reduced survival.^[75] Similarly, in breast cancer, higher pretreatment serum TK1 concentrations (often measured in U/L or pM) are associated with increased risk of recurrence and poorer response to neoadjuvant therapy, with levels typically dropping significantly in responders post-treatment.^[76]^[77] A cutoff value above 2.0 pM for serum TK1 peptide concentration has been associated with elevated risk for tumor proliferation in breast cancer, though assay-specific variations exist.^[77] Positron emission tomography (PET) imaging exploits TK1's role in nucleotide salvage by using 18F-fluorothymidine (18F-FLT), a radiolabeled thymidine analog that is phosphorylated and trapped in proliferating cells. This technique enables non-invasive detection of tumors with high TK1 activity, particularly in solid malignancies, achieving sensitivities of 80-90% for lesions in lung, breast, and esophageal cancers.^[78] For example, in non-small cell lung cancer, 18F-FLT PET demonstrates 90% sensitivity and 100% specificity when using standardized uptake value cutoffs, outperforming FDG-PET in distinguishing proliferative tumors from inflammatory lesions.^[78] In biotechnology, TK1 is integral to hybridoma selection protocols using HAT (hypoxanthine-aminopterin-thymidine) medium, which selectively eliminates unfused TK-deficient myeloma cells by blocking de novo nucleotide synthesis and relying on the salvage pathway. Aminopterin inhibits folate-dependent pathways, forcing cells to use hypoxanthine and thymidine via enzymes like hypoxanthine-guanine phosphoribosyltransferase and TK1; only hybridomas inheriting TK1 from fused B cells survive, enabling production of monoclonal antibodies for diagnostic assays.^[79] For viral infections, herpes simplex virus (HSV) TK assays facilitate monitoring of latency and antiviral resistance by phenotypically characterizing TK gene expression from clinical samples. These assays amplify and translate HSV-TK DNA in vitro to assess enzyme activity and size, identifying TK-negative mutants (prevalent in 95-96% of acyclovir-resistant strains) within two days, aiding rapid diagnosis in immunocompromised patients with latent or reactivating infections.^[80] Thymidine kinase in parasitic infections, such as Giardia intestinalis, has been characterized in 2022 studies as a high-affinity enzyme essential for DNA synthesis, highlighting its potential utility in assays for detecting and monitoring proliferative stages of the parasite.^[37] In clinical chemistry for hematologic cancers, the TK1 index—derived from serum activity levels—serves as a prognostic tool in acute myeloid leukemia (AML), where elevated values post-chemotherapy (>10 U/L) signal treatment failure, remission relapse, and diminished survival rates.^[3] This marker complements other proliferation indices to guide risk stratification and follow-up monitoring.^[81]

Therapeutic Applications

Thymidine kinase (TK), particularly the viral isoform from herpes simplex virus (HSV), plays a central role in antiviral therapies by selectively phosphorylating nucleoside analogs such as acyclovir (ACV) and ganciclovir (GCV) in infected cells. The viral TK converts these prodrugs to their monophosphate forms, after which host cellular kinases further phosphorylate them to triphosphates that inhibit viral DNA polymerase, halting replication. This mechanism underpins standard treatments for herpes zoster and other herpesvirus infections, where HSV-TK's higher affinity for ACV and GCV compared to human TK minimizes toxicity to uninfected cells.^[82] In suicide gene therapy, HSV-TK is engineered into therapeutic cells to enable controlled elimination via GCV administration. For instance, Zalmoxis, which expresses a modified HSV-TK (HSV-TK Mut2) in donor T-lymphocytes, was conditionally approved by the European Medicines Agency in 2016 for haploidentical hematopoietic stem cell transplantation in high-risk hematological malignancies like leukemia, allowing selective depletion of alloreactive T-cells to manage graft-versus-host disease. Although Zalmoxis was withdrawn in 2019 following suboptimal phase III results, ongoing clinical trials continue to explore HSV-TK/GCV systems, including exosome-mediated delivery for solid tumors such as Ewing sarcoma and glioblastoma, demonstrating safety and antitumor efficacy in phase I/II studies as of 2024-2025.^[83]^[84]^[85] Cytostatic agents like zidovudine (AZT), used in HIV treatment, rely on human TK1 for initial phosphorylation to its active triphosphate form, which inhibits reverse transcriptase and terminates viral DNA chain elongation. However, AZT's inhibition of mitochondrial TK2 contributes to toxicity, including myopathy and lactic acidosis, limiting long-term use. Additionally, elevated serum TK1 levels serve as a proliferation marker to predict chemotherapy response in solid tumors, with decreases post-treatment correlating to better outcomes in breast and colorectal cancers.^[86]^[87] Boron neutron capture therapy (BNCT) leverages TK to deliver boron-10 via TK-substrate analogs like boronated thymidine, which proliferating glioma cells selectively uptake and phosphorylate. Upon neutron irradiation, boron-10 captures neutrons to produce high-energy alpha particles, inducing localized cell death while sparing surrounding tissue; preclinical studies in EGFRvIII-positive glioma models confirm TK1's role in enhancing boron accumulation for targeted therapy.^[88]^[89] In antiparasitic applications, TK inhibitors target protozoan salvage pathways; for Trypanosoma brucei, the tandem TK structure enables efficient substrate binding, positioning it as a promising drug target for human African trypanosomiasis, with structural studies guiding inhibitor design. Giardia intestinalis TK, a high-affinity enzyme essential for DNA synthesis, has emerged as a 2020s drug target, where analogs like AZT inhibit parasite growth and cyst formation in vitro without significant host toxicity. Although Plasmodium falciparum lacks a canonical TK and relies on de novo synthesis, related thymidylate kinase inhibitors disrupt pyrimidine metabolism as antimalarial leads.^[90]^[37] Recent advances in TK2 deficiency, a mitochondrial disorder causing myopathy and DNA depletion, include pyrimidine nucleoside supplementation with thymidine and deoxycytidine to bypass enzymatic defects and restore mitochondrial function. A phase I/II trial (NCT03639701) demonstrated tolerability and motor improvements, leading to FDA approval of Kygevvi (doxecitine and doxribtimine combination) on November 3, 2025, with an approximately 86% reduced risk of death from treatment start (95% CI: 61%, 96%), and over 90% for patients whose symptoms began at age 12 or younger.^[91]^[92]

Measurement Methods

In Biological Fluids and Serum

Thymidine kinase (TK) activity and levels in biological fluids such as serum, plasma, and urine are primarily assessed through enzymatic assays that measure the phosphorylation of thymidine substrates, providing insights into systemic cell proliferation and turnover. The traditional DEAE-filter paper assay, a radiochemical method, involves incubating samples with [³H]-labeled thymidine and ATP, followed by spotting aliquots onto DEAE-cellulose filter paper (e.g., DE-81 disks) to separate the phosphorylated product (thymidine monophosphate) from unreacted substrate via washing in ammonium formate. This assay quantifies TK activity in units per liter (U/L), where 1 U/L corresponds to the phosphorylation of 1 nmol of thymidine per hour at 37°C, and has been widely used for serum TK1 evaluation due to its high sensitivity for detecting low-level activity. Complementing this, enzyme-linked immunosorbent assays (ELISAs) target TK1 protein directly; for instance, the AroCell TK 210 ELISA, a sandwich immunoassay, detects cytosolic TK1 with a sensitivity of 0.12 ng/mL, enabling quantification of the enzyme mass in serum without reliance on catalytic activity.^[93] These methods are applicable to urine, though serum remains the preferred matrix for routine analysis. Sample preparation for TK assays prioritizes serum over plasma to minimize interference from anticoagulants like heparin or EDTA, which can inhibit enzymatic reactions or bind substrates. Blood is collected in plain tubes, allowed to clot at room temperature for 30-60 minutes, and centrifuged at 1000-2000 × g for 10 minutes to obtain serum; hemolyzed or lipemic samples are discarded to avoid false elevations. TK1 in serum demonstrates stability for up to 24 hours when stored at 4°C, beyond which freezing at -20°C or lower is recommended to preserve activity, with repeated freeze-thaw cycles avoided to prevent degradation. In healthy individuals, serum TK activity typically falls below 5-10 U/L, reflecting basal cell turnover, while elevations to 10-100 U/L or higher are observed in malignancies such as leukemia, where rapid neoplastic proliferation releases TK1 into circulation. For example, in acute lymphoblastic leukemia, pretreatment levels often exceed 400 U/L median, correlating with disease burden. Standardization efforts align with general clinical enzymology principles, though specific IFCC reference procedures for TK remain limited; commercial immunoassay kits, such as automated chemiluminescent platforms, facilitate reproducibility across laboratories by using calibrated recombinant TK1 standards. As a tumor marker for monitoring treatment response, serum TK levels can briefly indicate proliferation dynamics in cancers like breast or lymphoma. Key limitations of fluid-based TK assays include non-specificity, as elevated levels may arise from TK1 release during cell death in non-malignant conditions like inflammation or regeneration, rather than solely active proliferation. Recent advancements, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), address this by quantifying deoxythymidine metabolites (e.g., deoxythymidine monophosphate) or using alternative substrates like 3'-azido-3'-deoxythymidine for indirect TK activity measurement, offering higher specificity and reduced reliance on radioactivity.

In Tissues and Cells

Thymidine kinase (TK) detection in tissues and cells relies on a variety of biochemical and imaging techniques tailored to fixed or live samples, enabling assessment of enzyme activity, protein expression, and localization. Biochemical assays typically begin with tissue homogenization to extract cellular contents, followed by measurement of TK enzymatic activity. The DE81 filter binding assay is a standard radiometric method that captures phosphorylated thymidine products on phosphocellulose filters after incubation with radiolabeled thymidine and ATP, allowing quantification of TK1 or TK2 activity in cell lysates or homogenates from various tissues.^[94] Western blotting complements this by detecting TK isozymes (TK1 and TK2) using isoform-specific antibodies on protein extracts from cultured cells or tissue samples, providing insights into relative expression levels and post-translational modifications.^[3] Immunohistochemistry (IHC) is widely used for visualizing TK in fixed tissue sections, particularly TK1, which serves as a proliferation marker. Antibodies targeting TK1 are applied to paraffin-embedded tumor samples, often in conjunction with Ki-67 staining to co-localize proliferating cells; semi-quantitative scoring of TK1 positivity yields a proliferation index that correlates with tumor aggressiveness, as seen in lung adenocarcinomas where TK1 outperforms Ki-67 in prognostic value.^[95] This approach highlights spatial distribution within tissue architecture, aiding in the identification of high-proliferative regions.^[96] For live-cell analysis, fluorescent reporters such as green fluorescent protein (GFP) fused to TK1 enable real-time imaging of enzyme dynamics during the cell cycle. These fusions localize primarily to the cytoplasm in S-phase cells, allowing visualization of TK1 upregulation and activity peaks via confocal microscopy in cultured mammalian cells.^[97] Flow cytometry facilitates high-throughput intracellular TK detection in cell suspensions from tissues or cultures. Permeabilization followed by staining with anti-TK1 antibodies permits quantification of enzyme levels in specific populations, such as CD34+ hematopoietic progenitors, where elevated TK1 indicates proliferative status.^[98] Recent methodological advances include CRISPR-Cas9-based TK knockout in cell lines and tissues, with validation typically via western blot to confirm absence of TK1 or TK2 protein, enabling functional studies of enzyme deficiency.^[99]

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