title: Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogen Candida albicans.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Cambridge, UK.
authors.forename: H M
authors.initials: HM
authors.lastname: Alloush
authors.forename: D
authors.initials: D
authors.lastname: Kerridge
mesh_terms: D002176:Candida albicans / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D010430:Pentosyltransferases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D008274:Magnesium