Evidence for multiple active states of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.
The lack of de novo purine biosynthesis in the malaria parasite Plasmodium falciparum makes the purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) a drug target. However, high activities for the purified recombinant enzyme have not been achieved, indicating that the P. falciparum enzyme requires very precise conditions for its maximal activity. In this report we have standardized the activation conditions necessary for high levels of activity, which is critically dependent on the ratios of enzyme, phosphoribosylpyrophosphate (PRPP), hypoxanthine, and buffer conditions. We demonstrate that excess substrates will push the enzyme to a less active state. We also present evidence for the existence of different kinetic states of the enzyme during activation and storage. Our kinetic data show that hypoxanthine is the substrate with highest affinity for the enzyme with a K(m) well below 1 microM. The activated enzyme has a maximum activity of 8.370 micromol/min/mg for hypoxanthine which is 10.8 times more than the previous reports. We discuss the biological relevance and implications of these results on drug design efforts.
Sujay Subbayya
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Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, 560 064, India.
Balaram
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D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006147:Guanine / Q000378:metabolism; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D010430:Pentosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D019271:Hypoxanthine; D006147:Guanine; D010754:Phosphoribosyl Pyrophosphate; D010430:Pentosyltransferases; C083867:hypoxanthine-guanine-xanthine phosphoribosyltransferase