Enzyme Activity Measurement for Thymidine Kinase

Creative Enzymes offers the most advanced spectrophotometric assays to measure the enzymatic activity of thymidine kinase accurately. We are able to work together with customers through the whole period of solving specific problems, mirroring target processes, and developing new methods. Working closely with our customers is the basis of our corporate success, and many customers have been loyal to us for many years.

Thymidine kinases (EC 2.7.1.21; formerly listed as EC 2.7.1.75; TKs) are essential enzymes for the initiation of the pyrimidine salvage pathway as they catalyze the magnesium dependent transfer of the γ-phosphate of ATP to thymidine (dThd), thus forming thymidine monophosphate (dTMP). dTMP is phosphorylated further by cellular enzymes to 2'-deoxythymidine triphosphate (dTTP), which serves as a substrate for DNA polymerase during replication. Hence thymidine kinases regulate the dTTP pool and are important regulatory enzymes for DNA biosynthesis. Thymidine kinases in mammals can be divided into two isozymes. TK1, a cytosolic enzyme, has a narrow specificity, phosphorylates only deoxythymidine and deoxyuridine, and is strictly cell-cycle-dependent. TK1 activity is low or absent in nondividing cells, starts to occur in late G1 cells, increases in S phase which is in close correlation to the increase in DNA synthesis, and disappears rapidly during mitosis. This fluctuation is critical for providing a balanced supply of dTTP for DNA replication. TK2 is a mitochondrial enzyme and cell cycle-independent.

In addition, thymidine kinases are classified into two subgroups according to their sequence and substrate specificity. Type I thymidine kinases include herpes simplex virus TK (HSV-TK) and mitochondrial human TK2. The much more widely spread type II thymidine kinases can be found in most organisms: eukaryotes, bacteria, and many viruses such as cowpox, variola, vaccinia, and poxviridae. These various viruses are likely targets for therapeutic intervention. Therefore, thymidine kinase plays a critical role in the activation of deoxynucleoside prodrugs used in antiviral and anticancer chemotherapy. For instance, the ability of type II TK to activate prodrugs is already documented for HIV therapy.

Thus, to further pursue the functional significance of thymidine kinases thoroughly, it is important to be able to monitor the activity of thymidine kinases. Creative Enzymes has the full capacity of the reliable enzymatic assays for thymidine kinases. For details, the activity of thymidine kinase could be determined by a coupled spectrophotometric assay, where the production of ADP is coupled to the production of NADH via pyruvate kinase/lactate dehydrogenase reactions and monitored at 340 nm. The products of Creative Enzymes are the most trusted and proven in the global market, resulting in high-quality data addressing customer’s needs. With scientific know-how and technical support from Creative Enzymes, your products will be positioned at a strong market presence.

Figure: The crystal structure of human thymidine kinase 1.
PDB: 1XBT