Inhibition and Mechanism of Plasmodium falciparum Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase

Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) is essential for purine salvage of hypoxanthine into parasite purine nucleotides. Transition state analogue inhibitors of PfHGXPRT are characterized by kinetic analysis, thermodynamic parameters, and X-ray crystal structures. Compound 1, 9-deazaguanine linked to an acyclic ribocation phosphonate mimic, shows a kinetic Ki of 0.5 nM. Isothermal titration calorimetry (ITC) experiments of 1 binding to PfHGXPRT reveal enthalpically driven binding with negative cooperativity for the binding of two inhibitor molecules in the tetrameric enzyme. Crystal structures of 1 bound to PfHGXPRT define the hydrogen bond and ionic contacts to complement binding thermodynamics. Dynamics of ribosyl transfer from 5-phospho-alpha-D-ribosyl 1-pyrophosphate (PRPP) to hypoxanthine were examined by 18O isotope exchange at the bridging phosphoryl oxygen of PRPP pyrophosphate. Rotational constraints or short transition state lifetimes prevent torsional rotation and positional isotope exchange of bridging to nonbridging oxygen in the alpha-pyrophosphoryl group. Thermodynamic analysis of the transition state analogue and magnesium pyrophosphate binding reveal random and cooperative binding to PfHGXPRT, unlike the obligatory ordered reaction kinetics reported earlier for substrate kinetics.

Automated summary

Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) is an essential enzyme for the salvage pathway of hypoxanthine into parasite purine nucleotides. In this study, transition state analogue inhibitors of PfHGXPRT were characterized using kinetic analysis, thermodynamic parameters, and X-ray crystal structures. Compound 1, which is an acyclic ribocation phosphonate mimic linked to 9-deazaguanine, showed a kinetic Ki of 0.5 nM. Isothermal titration calorimetry experiments revealed enthalpically driven binding of compound 1 to PfHGXPRT with negative cooperativity. Crystal structures of the inhibitor bound to the enzyme provided insights into the hydrogen bond and ionic contacts involved in the binding process. The dynamics of ribosyl transfer from 5-phospho-alpha-D-ribosyl 1-pyrophosphate (PRPP) to hypoxanthine were investigated using 18O isotope exchange, which showed that rotational constraints and short transition state lifetimes prevented positional isotope exchange. The thermodynamic analysis of the transition state analogue and magnesium pyrophosphate binding indicated random and cooperative binding to PfHGXPRT, suggesting a different mechanism from the previously reported substrate kinetics.