The hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus has unique properties.
Tritrichomonas foetus, an anaerobic, flagellated protozoan parasite, is incapable of de novo purine nucleotide synthesis, and depends primarily on the salvage of purine bases from the host. The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from this organism has been purified to homogeneity by ammonium sulfate precipitation and Sephacryl-HR100 gel filtration, followed by anion exchange FPLC. Hypoxanthine, guanine and xanthine phosphoribosyltransferase activities co-eluted in all the purification steps, suggesting that they are associated with the same enzyme protein. The molecular mass of the native protein, as estimated by gel filtration, is 24 kDa. The molecular mass estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is also 24 kDa. Non-denaturing polyacrylamide gel electrophoresis of the purified protein, followed by activity staining with either [14C]hypoxanthine, [14C]guanine or [14C]xanthine, also demonstrates that the enzyme is a monomer of 24 kDa. This monomeric structure is distinctive from all the other reported PRTases which are either dimers or tetramers. Furthermore, unlike the mammalian HGPRTase, which is heat stable, the T. foetus enzyme is heat labile. Kinetic studies with the purified T. foetus HGXPRTase showed that the apparent Kms for hypoxanthine, guanine and xanthine were 4.1 microM, 3.8 microM and 52.4 microM respectively. This recognition of xanthine as a substrate by the parasite enzyme with only about a 10-fold higher Km value than those for hypoxanthine and guanine distinguishes it from the mammalian HGPRTase, which cannot use xanthine as a substrate, as well as the HGXPRTases of Eimeria tenella and Plasmodium falciparum, which are dimers, with xanthine about 100-times less proficient as a substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Beck
J T
JT
Department of Pharmaceutical Chemistry, University of California San Francisco 94143.
Wang
C C
CC
D000818:Animals; D002412:Cations / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D016835:Tritrichomonas foetus / Q000201:enzymology*; D019820:Xanthine; D014970:Xanthines
D002412:Cations; D014970:Xanthines; D019820:Xanthine; D010430:Pentosyltransferases; C083867:hypoxanthine-guanine-xanthine phosphoribosyltransferase