Fish liver pyrimidine deoxynucleoside phosphorylase and deoxyribosyltransferase

Abstract

A pyrimidine deoxynucleoside phosphorylase, which also possessed deoxyribosyltransferase activity, was purified about 89-fold from trout liver with an average recovery of 67%. Starch-gel electrophoresis showed that four proteins were present, one of which possessed both activities. The phosphorylase utilized uracil, thymine, and 5-halogen-substituted uracils, but not cytosine, adenine, or hypoxanthine in presence of deoxyribose 1-phosphate. Deoxyuridine, thymidine, and the deoxynucleosides of the 5-halogen-substituted uracils, but not uridine, deoxycytidine, deoxyadenosine, deoxyinosine, or deoxyguanosine, were substrates for the reverse reaction. The enzyme possessed a much higher affinity for deoxyuridine than for thymidine, the Km values being 1.7 × 10−3 M and 1 × 10−2 M respectively. The optimum pH was 6.2. The enzyme was destroyed by freezing, and was rapidly inactivated at 45° and by dialysis. It was very unstable in absence of 2-mercaptoethanol and fairly unstable in absence of orthophosphate. Deoxyuridine phosphorolysis was inhibited noncompetitively by thymine and uracil. The transferase exhibited no sharp pH optimum and utilized the same substrates as did the phosphorylase. The ratio transferase/phosphorylase remained approximately constant (4.0) during purification. The enzyme formed 3-deoxyribosylxanthine in small amounts from xanthine plus deoxyribose 1-phosphate and from deoxyuridine plus xanthine.