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Enzyme Activity Measurement for Thymidine Phosphorylase Using Spectrophotometric Assays



Creative Enzymes is an industrial biotech company specialized in enzyme activity assays. We firmly believe that the quality of our services is the basic requirement for our corporate success and the basis of our high-level customer satisfaction. The professional techniques and high-quality services of Creative Enzymes support global customers in various activity assays for transferases, including thymidine phosphorylase.

Thymidine phosphorylase (EC 2.4.2.4; TP) is a unique metabolic control enzyme which catalyzes the reversible phosphorolysis of thymidine, deoxyuridine, and their analogs to their respective bases and 2-deoxyribose-1-phosphate. It also catalyzes deoxyribosyl transfer from one deoxynucleoside to another base to form a second nucleoside. Thymidine phosphorylase is a dimeric enzyme made up of two identical subunits, the molecular mass of each being about 55,000 daltons in mammals. Human thymidine phosphorylase shares 39% of its sequence identity with Escherichia coli thymidine phosphorylase giving rise to significant sequence homology.

Figure 1: Phosphorolysis reaction of thymidine with thymidine phosphorylase. Figure 1: Phosphorolysis reaction of thymidine with thymidine phosphorylase.
Reference: Hatano A, et al. Bioorganic & Medicinal Chemistry, 2008, 16(7):3866-70.

The mechanism of the phosphorolysis reaction is considered as a nucleophilic process: the ribosyl 1-anomeric carbon undergoes addition of a phosphate ion at its α face. It was reported that the crystal structure of thymidine phosphorylase complexed with TPI (5-chloro-6-[1-(2-iminopyrrolidinyl) methyl]uracil) HCl in an active conformation, which further confirms the mechanisms through this inhibited complex.

Thymidine phosphorylase is identical with platelet-derived endothelial cell growth factor (PD-ECGF), and the enzymatic activity of thymidine phosphorylase is required for angiogenesis. Thus, thymidine phosphorylase is a good target for the development of anti-tumor drugs. Characterizing the catalytic behavior of thymidine phosphorylase is vital for the fundamental importance and serves as the first step to the design of new anti-tumor agents.

Having tested activities of numerous enzymes in the past few years, Creative Enzymes has accumulated extensive and professional experiences, which can provide rapid and utmost-quality assay services for thymidine phosphorylase. Enzymatic activity of thymidine phosphorylase can be determined spectrophotometrically at 25°C by continuously measuring the difference in absorption of a nucleotide at 290 nm. Fully equipped with the most advanced spectrophotometric instruments, we assure the reliability of our tests.

Figure 2: The crystal structure of thymidine phosphorylase from E.coli.
Figure 2: The crystal structure of thymidine phosphorylase from E.coli.
PDB: 4EAF



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