“…Figure K shows thymidine kinase, a key enzyme in the synthesis of thymidine monophosphate, and Figure J shows hexokinase, an enzyme that catalyzes the phosphorylation of glucose into glucose-6-phosphate. Thymidine kinase has been reported to assemble as a low activity dimer and as a highly active tetramer . Hexokinase forms dimers in solution .…”
Section: Resultsmentioning
confidence: 99%
“…Thymidine kinase has been reported to assemble as a low activity dimer and as a highly active tetramer. 28 Hexokinase forms dimers in solution. 29 Interestingly, both X-ray crystal structures show a typical string of stacked arginines with C ζ −C ζ distances <5 Å.…”
Protein-protein interfaces are often stabilized by a small number of dominant contacts, exemplified by the overrepresentation of arginine residues at oligomerization interfaces. Positively charged arginines are most commonly involved in ion pairs of opposite charge; however, previous work of Scheraga and coworkers described the stable, close range interaction between guanidinium pairs in a solvated environment. To extend this work, we searched over 70 thousand protein structures and complexes for unusual formations of arginine residues supported by the electron density. Symmetry transformations were used to generate full assemblies. Clusters of four to eight arginine residues with Cζ-Cζ distances < 5 Å, organized as rings with 4 to 8 members, stacks of two arginines, and strings of stacked arginines, are commonly located at the interfaces of oligomeric proteins. The positive charge is properly balanced by negatively charged counter ions in about 90% of the cases. We also observed planar stacking of guanidinium groups, bridged by hydrogen bonds and interactions with water molecules. The guanidinium groups are commonly involved in 5 hydrogen bonds with water molecules and acceptor groups from surrounding amino acids. Water molecules have a bridging effect on the arginine pairs, but in some cases small molecular weight chemicals in the crystallization buffer may be misinterpreted as water molecules. In summary, despite electrostatic repulsion, arginines do form various clusters that are exposed to interact with and potentially be controlled or switched by charged metabolites, membrane lipids, nucleic acids or side chains of other proteins. Control of the stability of arginine clusters may play an important role in protein-protein oligomerization, molecular recognition and ligand binding.
“…Figure K shows thymidine kinase, a key enzyme in the synthesis of thymidine monophosphate, and Figure J shows hexokinase, an enzyme that catalyzes the phosphorylation of glucose into glucose-6-phosphate. Thymidine kinase has been reported to assemble as a low activity dimer and as a highly active tetramer . Hexokinase forms dimers in solution .…”
Section: Resultsmentioning
confidence: 99%
“…Thymidine kinase has been reported to assemble as a low activity dimer and as a highly active tetramer. 28 Hexokinase forms dimers in solution. 29 Interestingly, both X-ray crystal structures show a typical string of stacked arginines with C ζ −C ζ distances <5 Å.…”
Protein-protein interfaces are often stabilized by a small number of dominant contacts, exemplified by the overrepresentation of arginine residues at oligomerization interfaces. Positively charged arginines are most commonly involved in ion pairs of opposite charge; however, previous work of Scheraga and coworkers described the stable, close range interaction between guanidinium pairs in a solvated environment. To extend this work, we searched over 70 thousand protein structures and complexes for unusual formations of arginine residues supported by the electron density. Symmetry transformations were used to generate full assemblies. Clusters of four to eight arginine residues with Cζ-Cζ distances < 5 Å, organized as rings with 4 to 8 members, stacks of two arginines, and strings of stacked arginines, are commonly located at the interfaces of oligomeric proteins. The positive charge is properly balanced by negatively charged counter ions in about 90% of the cases. We also observed planar stacking of guanidinium groups, bridged by hydrogen bonds and interactions with water molecules. The guanidinium groups are commonly involved in 5 hydrogen bonds with water molecules and acceptor groups from surrounding amino acids. Water molecules have a bridging effect on the arginine pairs, but in some cases small molecular weight chemicals in the crystallization buffer may be misinterpreted as water molecules. In summary, despite electrostatic repulsion, arginines do form various clusters that are exposed to interact with and potentially be controlled or switched by charged metabolites, membrane lipids, nucleic acids or side chains of other proteins. Control of the stability of arginine clusters may play an important role in protein-protein oligomerization, molecular recognition and ligand binding.
“…The K M and V max values for ATP were 1501 μM and 3.00 μmol/min/mg, respectively (Table 3 ). These results suggest that the binding of ATP may affect subunit interactions, as observed in human TK1 [ 22 ]. Fig.…”
Background
Thymidine kinase 1 (TK1) plays a key role in the synthesis of deoxythymidine triphosphate (dTTP) and is thus important for DNA replication and cell proliferation. The expression of TK1 is highest during S-phase, and it is rapidly degraded after mitosis. In cancer cells, TK1 is upregulated, resulting in leakage of excess TK1 into the blood. Consequently, serum TK1 has been used as a diagnostic and prognostic cancer biomarker, mainly in human medicine. The aims of this work were to characterize equine TK1 and to evaluate its suitability as a serum biomarker for equine lymphoma.
Results
Equine TK1 was cloned, expressed in E. coli and affinity purified. The purified recombinant horse TK1 showed broad substrate specificity, phosphorylating pyrimidine deoxyribo- and ribonucleosides and, to some extent, purine deoxynucleosides, including anticancer and antiviral nucleoside analogues. ATP was the preferred phosphate donor. Serum TK1 activity was measured in samples collected from horses with confirmed or suspected lymphoma and control horses with and without concurrent diseases. Serum TK1 activity levels were significantly higher in horses with lymphoma (p < 0.0005) and suspected lymphoma (p < 0.02) and in tumour-free groups with diverse diseases (p < 0.03) than in controls without concurrent diseases. There was a significant difference between the lymphoma group and the tumour-free group with diverse diseases (p < 0.0006). Furthermore, receiver operating characteristic analysis revealed a sensitivity of 0.86, a specificity of 0.95 and an AUC (area under the curve) of 0.92 compared to the controls without concurrent diseases, with a sensitivity of 0.97, a specificity of 0.71 and an AUC of 0.88 when compared with the tumour-free group with diverse diseases.
Conclusion
Equine TK1 showed high specific activity and broader substrate specificity than human TK1. Anticancer and antiviral thymidine analogues were efficiently phosphorylated by horse TK1, suggesting that these analogues might be good candidates for chemotherapy in horses. Serum TK1 activity was significantly higher in horses with lymphoma than in controls. ROC analysis indicated that serum TK1 could serve as a promising cancer biomarker in horses.
“…The K M and V max values for ATP were 794 μM and 2.38 μmol/min/mg respectively (Table 3 ). These results suggest that the binding of ATP may affect subunit interactions as seen in cases of human TK1, i.e., at higher ATP concentrations the enzyme is in a tetramer form with higher catalytic activity, a typical behavior of a cooperative or allosteric enzyme [ 24 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…However, with human native TK1, the phosphorylation of AZT followed the Michaelis-Menten kinetics [ 23 , 25 ]. The ATP kinetics showed positive cooperativity, suggesting that ATP binding affected subunit interactions, similar to human TK1, which showed a concentration dependent transition between the tetramer form with a higher catalytic activity and the dimer form with a lower catalytic activity [ 24 ].…”
Background
Thymidine kinase 1 (TK1) catalyzes the initial phosphorylation of thymidine in the salvage pathway synthesis of dTTP, an essential building block of DNA. TK1 is a cytosolic enzyme with its highest level during the S-phase of the cell cycle. In cancer cells TK1 is upregulated and excess TK1 is leaked into the blood. Therefore, serum TK1 has been used as biomarker for cancer diagnosis and prognosis in human medicine. Feline TK1 shows high sequence similarity to TK1 from other species. The aim of this study was to characterize feline TK1 and evaluate if serum TK1 can be used as a diagnostic biomarker.
Results
Feline TK1 was cloned, expressed and affinity purified. The purified feline TK1 phosphorylated not only pyrimidine deoxyribonucleosides but also pyrimidine ribonucleosides and to some extent purine deoxynucleosides. A number of anticancer and antiviral nucleoside analogs also served as substrates with fairly high efficiency. ATP and dATP were the preferred phosphate donor. Serum TK1 activity in felines with malignant diseases was significantly higher than that in healthy individuals. ROC analysis revealed an area under the curve (AUC) of 0.98 with a sensitivity of 0.83 and a specificity of 0.95 for felines with lymphoma. Serum TK1 activity in felines with IBD or inflammatory disease was within the same range as healthy ones. Furthermore, in felines with lymphoma serum TK1 activity returned to normal levels in response to treatment.
Conclusion
Feline TK1 has high specific activity and a broader substrate specificity in comparison with TK1 from other species. Serum TK1 activity in felines with malignant diseases is significantly higher than that in normal felines and in felines with inflammatory diseases. These results suggest that serum TK1 may be a promising biomarker for the diagnosis and monitoring of malignant diseases and for the differential diagnosis of certain inflammatory disease.
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