CHARACTERIZATION OF A PARTIALLY PURIFIED URACIL PHOSPHORIBOSYLTRANSFERASE FROM THE OPPORTUNISTIC PATHOGEN CANDIDA-ALBICANS

This paper describes for the first time the partial purification and p roperties of uracil phosphoribosyltransferase (UPRTase) from the yeast Candida albicans. UPRTase was purified 38 fold by acid precipitation, DEAE-Sephacel chromatography and ultrafiltration. Further purificatio n of UPRTase was unsuccessful due to the labile nature of the enzyme a nd the failure in obtaining satisfactory stabilizing conditions, SDS-P AGE suggested that the enzyme exists as a dimer of two dissimilar subu nits with molecular masses of 47 and 38 kDa. The pH optimum for phosph oribosylation was about 7.5 and the optimal Mg++ concentration was 2 m M. The kinetics of the enzymes for its substrates, uracil and 5-phosph oribosyl-1-pyrophosphate (PRPP) were determined by measuring initial e nzyme velocities over a wide range of concentrations of either substra te 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 phosphoribosyltransfe rases. The enzyme has a low affinity for its substrates (K-m = 70.5 an d 186 mu M for uracil and PRPP, respectively) as compared with those o f E. coli and baker's yeast. Inhibition studies indicate that 5-fluoro uracil acts as an alternative substrate for UPRTase with 1.6 times hig her specific activity.