Pentosyl Transfer Mechanisms of the Mammalian Nucleoside Phosphorylases

Phosphate dependence obeying Michaelis-Menten type kinetics was found to be a common feature of pentosyl transfer catalyzed by guinea pig uridine phosphorylase and rabbit thymidine phosphorylase. Despite this basic similarity, significant differences in the characteristics of this phosphate dependence were observed. Stoichiometric amounts of phosphate (Km' = 3 x 10-4 m) were required for optimal rates of pentosyl transfer catalyzed by uridine phosphorylase, while less than stoichiometric amounts (Km' = 3 x 10-5 m) were required with thymidine phosphorylase. Sephadex G-100 chromatography essentially eliminated the pentosyl transfer activity of the uridine phosphorylase preparation in the absence of added phosphate. However, after the same treatment, thymidine phosphorylase retained a significant amount of transfer activity in the absence of added phosphate. Relatively high concentrations of phosphate inhibited pentosyl transfer catalyzed by uridine phosphorylase, but did not inhibit the transfer activity of thymidine phosphorylase. These results suggest an ordered mechanism for uridine phosphorylase, similar to that of purine nucleoside phosphorylase, involving the initial addition of pyrimidine or nucleoside to the free enzyme. Pentosyl transfer by this mechanism involves a free pentose 1-phosphate intermediate. For thymidine phosphorylase, the available data suggest an ordered mechanism involving the reverse sequence of addition, i.e. the initial addition of phosphate or pentose 1-phosphate to the free enzyme. Pentosyl transfer by this mechanism involves a pentose 1-phosphate intermediate which is bound to the enzyme rather than being free. At relatively higher concentrations, arsenate substituted for the phosphate requirement of pentosyl transfer reactions catalyzed by uridine phosphorylase. The apparent Km value for arsenate was 3.6 x 10-4 m as compared with a value of 3 x 10-5 m for phosphate. Arsenate also substituted for phosphate in pentosyl transfer reactions catalyzed by thymidine phosphorylase. These findings suggest that the pentose 1-arsenate esters are sufficiently stable to act as intermediates in pentosyl transfer catalyzed by the pyrimidine nucleoside phosphorylases.