Thymidine phosphorylase (thymine: orthophosphate deoxyribosyl transferase, EC 2.4.2.4) is a cytosolic enzyme which catalyses the reversible phosphorolysis of thymidine and other pyrimidine 2'-deoxyribosides, except for 4-amino substituted compounds such as 2'-deoxycytidine, as follows: EQU PyrdR+P.sub.i.rarw..fwdarw.Pyr+dR-1-P
The phosphorolytic and synthetic reactions may also be used to transfer a deoxyribose moiety of one deoxynucleoside to form a second deoxynucleoside in a nucleoside deoxyribosyl transferase reaction (Schwartz, M. (1971) Eur. J. Biochem. 21, 191-198).
Thymidine phosphorylase has been purified and characterized from a number of micro-organisms (Schwartz, M. (1971) Eur. J. Biochem. 21, 191-198, Schwartz, M. (1978) Methods Enzymol. 51, 442-445; Avraham, Y. et al (1990) Biochim. Biophys. Acta 1040, 287-293; Hoffee, P. A. et al (1978) Methods Enzymol. 51, 437-442) and from human tissues (Desgranges, C. et al (1981) Biochim. Biophys. Acta. 654, 211-218; Kubilus, J. et al (1978) Biochim. Biophys. Acta 527, 221-228; Yoshimura et al (1990) Biochim. Biophys. Acta 1034, 107-113). Escherichia coli thymidine phosphorylase is a dimer of 90 kD composed of two identical subunits with a molecular weight of 45 kD (Schwartz, M. (1978) Methods Enzymol. 51, 442-445; Walter, M. et al (1990) J. Biol. Chem. 265, 14016-14022). The three-dimensional crystal structure of Escherichia coli has been determined to resolution of 2.8 .ANG. (Walter, M. et al (1990) J. Biol. Chem. 265, 14016-14022). The monomer subunit consists of a small .alpha.-helical domain and a large .alpha./.beta. domain. The active site, which binds both thymidine and phosphate, has been located in a cleft between the two domains.
Human thymidine phosphorylase has been identified in many tissues including lymphocytes, heart, spleen, lung and placenta (Yoshimura, A. et al (1990) Biochim. Biophys. Acta. 1034, 107-113) and purified from both placenta (Yoshimura, A. et al (1990) Biochim. Biophys. Acta. 1034, 107-113; Kubilus, J. (1978) Biochem. Biophys. Acta. 527, 221-228) and platelets (Desgranges, C. (1981) Biochim. Biophys. Acta 654, 211-218). It has been suggested that thymidine phosphorylase plays an essential role in maintaining intracellular thymidine homeostasis (Shaw, T. et al (1988) Mutant Res. 200, 99-116). The thymidine salvage pathway requires the action of a permease to transport thymidine across the lipid bilayer. Intracellular thymidine is then phosphorylated by thymidine kinase, generating thymidine monophosphate (TMP) which is further phosphorylated to generate thymidine triphosphate.
Thymidine triphosphate not only regulates the thymidine salvage pathway by inhibition of thymidine kinase, but also inhibits the production of other deoxyribonucleotides by allosteric effects on ribonucleotide diphosphate reductase. The action of thymidine phosphorylase may therefore be to regulate the size of the intracellular thymidine nucleotide pool and hence the size of the other nucleotide pools via ribonucleotide diphosphate reductase as suggested by Shaw, T. et al, Mutant Res. 200, 99-116 (1988), and hence regulate DNA synthesis.
There has been no suggestion that thymidine phosphorylase could be used as an extracellular growth factor in medicine.