Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogen Candida albicans.

This paper describes for the first time the partial purification and properties of uracil phosphoribosyltransferase (UPRTase) from the yeast Candida albicans. UPRTase was purified 38 fold by acid precipitation, DEAE-Sephacel chromatography and ultrafiltration. Further purification of UPRTase was unsuccessful due to the labile nature of the enzyme and the failure in obtaining satisfactory stabilizing conditions. SDS-PAGE suggested that the enzyme exists as a dimer of two dissimilar subunits with molecular masses of 47 and 38 kDa. The pH optimum for phosphoribosylation was about 7.5 and the optimal Mg++ concentration was 2 mM. The kinetics of the enzymes for its substrates, uracil and 5-phosphoribosyl-1-pyrophosphate (PRPP) were determined by measuring initial enzyme velocities over a wide range of concentrations of either substrate at different fixed concentrations of the second substrate. Graphic analysis of the data by Hanes-Woolf plots indicated that the reaction is indistinguishable from a double displacement reaction. 'Ping pong' mechanism has been previously reported for other phosphoribosyltransferases. The enzyme has a low affinity for its substrates (Km = 70.5 and 186 microM for uracil and PRPP, respectively) as compared with those of E. coli and baker's yeast. Inhibition studies indicate that 5-fluorouracil acts as an alternative substrate for UPRTase with 1.6 times higher specific activity.