Thymine nucleoside phosphorylase

A close look at this reaction reveals that in the opposite direction, the reaction is of the phosphorolysis type. For this reason, the enzymes catalyzing the reaction with ribose-l-phosphate are called phosphorylases, and they also participate in nucleic acid degradation pathways. Purine nucleoside phosphorylases thus convert hypoxanthine and guanine to either inosine and guanosine if ribose-l-phosphate is the substrate or to deoxyinosine and deoxyguanosine if deoxyribose-1-phosphate is the substrate. Uridine phosphorylase converts uracil to uridine in the presence of ribose-l-phosphate, and thymidine is formed from thymine and deoxyribose-l-phosphate through the action of thymidine phosphorylase. 

Uridine phosphorylase, but not thymidine phosphorylase, is inhibited powerfully by the thymine nucleosides, deoiQ xylosylthymine and deoi -glycosylthymine ( ) ... 

However, for adenine, guanine, and uracil, the dominant route of anabolism is by way of their ribonucleotide derivatives and traffic along the deoxyribosidic route is not ordinarily significant. Because cytosine is not a substrate for nucleoside phosphorylases, incorporation by the phos-phorylase-kinase route is not possible for this base. The other pyrimidine base of DNA, thymine, is poorly anabolized by both animal and bacterial cells, in spite of the fact that most cells possess thymidine phosphorylase, the action of which is readily reversible. This suggests that ordinarily cellular supplies of deoxyribose 1-phosphate are not available for base anabolism. Experiments are cited below in which it was demonstrated that a significant contribution to the biogenesis of deoxyribose of DNA in E. colt cells did not occur by a route other than ribonucleotide reduction. 

In addition to participation in the deoxyribosyl transfer reactions described above, in which free deoxyribose 1-phosphate is formed as an intermediate, thymidine phosphorylase also catalyzes deoxyribosyl transfers involving thymine and uracil in which deoxyribosyl phosphate is an intermediate but is enzyme-bound (18-20). Such transfers require non-stoichiometric amounts of phosphate (19). The reaction mechanisms of uridine phosphorylase and purine nucleoside phosphorylase are not of this type and, accordingly, direct deoxyribosyl transfers occur only between substrates for thymidine phosphorylase, as exemplified by reaction (5) above and the following (15) (the asterisk indicates C-labeling) ... 

The mechanism of formation of thymine and hydroxymethylcytosine are not yet known, but the addition of a carbon to position 5 appears to involve a transfer from a folic acid derivative. As in the case of the purines, the relation between ribose and deox3nibose nucleotides is obscure. Deoxyribonucleosides can be formed from deoxyribose-l-phosphate by a nucleoside phosphorylase reaction. 

Labeled compounds. Thymine-2- G and excess deoxyriboso-1-phosphate incubated 0.5 hr. at 37 with nucleoside phosphorylase from Escherichia coli B. in Tris buffer and Mg-chloride soln. thymidine-2- C. Y ca. 77%. F. e. s. J. Filip and Z. Nejedly, J. Labelled Compounds 3, 128 (1967). 

The bromide (2a) reacted smoothly with 2,4-diethoxy-5-methylpyrimi-dine to give, after de-ethylation and deacylation, l-(2-deoxy-/ -D-arabino-hexopyranosyl)thymine (3) (15). The new nucleoside (3) is the first truly competitive inhibitor of a pyrimidine phosphorylase (7), that is, it inhibits the phosphorylase, yet is not a substrate for the enzyme. It was recently shown that 3 enhances the incorporation of 2 -deoxy-5-iodouridine in vivo in cats (8). 

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