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 ph osphoribosyltransferase (HGXPRT) a drug target. However, high activities fo r the purified recombinant enzyme have not been achieved, indicating that t he P. falciparum enzyme requires very precise conditions for its maximal ac tivity. In this report we have standardized the activation conditions neces sary for high levels of activity, which is critically dependent on the rati os 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 differen t kinetic states of the enzyme during activation and storage. Our kinetic d ata show that hypoxanthine is the substrate with highest affinity for the e nzyme with a K-m well below 1 muM. The activated enzyme has a maximum activ ity of 8.370 mu mol/min/mg for hypoxanthine which is 10.8 times more than t he previous reports. We discuss the biological relevance and implications o f these results on drug design efforts. (C) 2000 Academic Press.