Approaching the transition state in the crystal structure of a phosphoribosyltransferase

Hypoxanthine phosphoribosyltransferase (HPRT) salvages 6-oxopurine bases in the nucleotide metabolic pathway. The 1.8 Angstrom crystal structure of an asymmetric dimer of the HPRT from the protozoan parasite Trypanosoma cruzi was determined in a ternary complex with the primary substrate phosphoribo sylpyrophosphate (PRPP) and an analogue of the substrate hypoxanthine, reve aling both open and closed active site conformations. The ligands are posit ioned for in-line nucleophilic attack at the PRPP ribose C1' by two metal i ons which straddle the pyrophosphate leaving group. The structure provides the first evidence for the involvement of two metal ions in the HPRT-cataly zed reaction, and structural details further suggest the mechanism may proc eed via S(N)2-type chemistry. The closed conformation reveals the structura l roles for invariant flexible loop residues Ser103 and Tyr104 and supports a role for the loop in the liberation of pyrophosphate. The pre-transition state structure is valuable for understanding the enzyme mechanism, as wel l as providing a foundation for antiparasite drug design efforts against T. cruzi, which causes Chagas' disease in humans. Additionally, the structure illuminates the molecular basis of three inherited mutations in the human HPRT leading to Lesch-Nyhan syndrome (D193N) or gout (S103R or S109L), as t he homologous residues in the trypanosomal enzyme contribute to the previou sly unrecognized Mg2+ ion binding site and to the formation of the closed f lexible loop, respectively.