Biochemical characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphorybosyltransferase: role of histidine residue in substrate selectivity.
The enzyme hypoxanthine-guanine phosphorybosyltransferase (HGPRT) in the malarial parasite Plasmodium falciparum (Pf) is central to the salvage pathway for purine nucleotide biosynthesis and is a potential antimalarial chemotherapeutic target. The pH profile of the enzyme activity using xanthine as a substrate shows the possible involvement of a histidine residue in the activity of the enzyme. Chemical modification studies using diethylpyrocarbonate (DEPC) also corroborate this hypothesis. A comparative sequence alignment of Pf HGPRT with the human, Tricomonus foetus and Toxoplasma gondii HGPRT, coupled with the 3D structural alignment between these enzymes indicated that a histidine residue at position 196 of the Pf HGPRT sequence was located in the close proximity to the active site. Site directed mutagenesis of this histidine residue to lysine (the corresponding residue in the human enzyme) specifically abrogated xanthine and guanine utilization of the enzyme without affecting the conversion of hypoxanthine to its corresponding nucleotide. The mechanism of action for this enzyme was evaluated by steady state kinetics for the substrates xanthine, guanine and PRPP and product inhibition studies. The results indicate the possibility of ping-pong mechanism for the enzyme in contrast to the ternary complex mechanism followed by the human enzyme. These results show that the difference in human and malarial HGPRT can be gainfully exploited to design specific inhibitor for this enzyme.
Sarkar
Dhiman
D
AstraZeneca R & D, Bellary Road, Hebbal, Bangalore 560024, India.
Ghosh
Indira
I
Datta
Santanu
S
D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; C437127:O,O-diethylphosphorylcholine iodide; D010767:Phosphorylcholine; D006639:Histidine; D010430:Pentosyltransferases; C083867:hypoxanthine-guanine-xanthine phosphoribosyltransferase