Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from <i>Plasmodium falciparum</i>

<i>Plasmodium falciparum</i> parasites are purine auxotrophs that rely exclusively on the salvage of preformed purines from their human hosts to supply the requirement for purine nucleotides. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) catalyzes the freely reversible Mg²⁺-dependent conversion of 6-oxopurine bases to their respective nucleotides and inorganic pyrophosphate. The phosphoribosyl group is derived from 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP). The enzyme from malaria parasites (<i>Pf</i>HGXPRT) is essential as hypoxanthine is the major precursor in purine metabolism. We used specific heavy atom labels in PRPP and hypoxanthine to measure primary (1-¹⁴C and 9-¹⁵N) and secondary (1-³H and 7-¹⁵N) intrinsic kinetic isotope effect (KIE) values for <i>Pf</i>HGXPRT. Intrinsic isotope effects contain information for understanding enzymatic transition state properties. The transition state of <i>Pf</i>HGXPRT was explored by matching KIE values predicted from quantum mechanical calculations to the intrinsic values determined experimentally. This approach provides information about <i>Pf</i>HGXPRT transition state bond lengths, geometry, and atomic charge distribution. The transition state structure of <i>Pf</i>HGXPRT was determined in the physiological direction of addition of ribose 5-phosphate to hypoxanthine by overcoming the chemical instability of PRPP. The transition state for <i>Pf</i>HGXPRT forms nucleotides through a well-developed and near-symmetrical D_N*A_N, S_N1-like transition state