Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme.
The human malaria parasite Plasmodium falciparum is auxotrophic for purines and relies on the purine salvage pathway for the synthesis of its purine nucleotides. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a key purine salvage enzyme in P. falciparum, making it a potential target for chemotherapy. Previous attempts to purify this enzyme have been unsuccessful because of the difficulty in obtaining cultured parasite material and because of the inherent instability of the enzyme during purification and storage. Other groups have tried to express recombinant P. falciparum HGXPRT but only small amounts of activity were obtained. The successful expression of recombinant P. falciparum HGXPRT in Escherichia coli has now been achieved and the enzyme purified to homogeneity in mg quantities. The measured molecular mass of 26 229+/-2 Da is in excellent agreement with the calculated value of 26232 Da. A method to stabilise the activity and to reactivate inactive samples has been developed. The subunit structure of P. Jilciparum HGXPRT has been determined by ultracentrifugation in the absence (tetramer) and presence (dimer) of KC1. Kinetic constants were determined for 5-phospho-alpha-D-ribosyl-1-pyrophosphate, for the three naturally-occurring 6-oxopurine bases guanine, hypoxanthine, and xanthine and for the base analogue, allopurinol. Differences in specificity between the purified P. falciparum HGXPRT and human hypoxanthine guanine phosphoribosyltransferase enzymes were detected which may be able to be exploited in rational drug design.
Keough
D T
DT
Centre for Protein Structure Function and Engineering, Department of Biochemistry, University of Queensland, Brisbane, Australia.
Ng
A L
AL
Winzor
D J
DJ
Emmerson
B T
BT
de Jersey
J
J
D000818:Animals; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D008627:Mercuric Chloride / Q000494:pharmacology; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity
D011994:Recombinant Proteins; D008627:Mercuric Chloride; D011189:Potassium Chloride; D010430:Pentosyltransferases; C083867:hypoxanthine-guanine-xanthine phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase