ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) (
ATP:
oxaloacetate carboxylyase (transphosphorylating), EC 4.1.1.49) is a key
enzyme involved in the catabolism of
glucose and
amino acids in the parasite Trypanosoma cruzi, the causative agent of
Chagas' disease. Due to the significant differences in the amino acid sequence and substrate specificity of the human
enzyme (PEPCK (
GTP-dependent), EC 4.1.1.32), the parasite
enzyme has been considered a good target for the development of new anti-chagasic drugs. We have solved the crystal structure of the recombinant PEPCK of T. cruzi up to 2.0 A resolution, characterised the dimeric organisation of the
enzyme by
solution small angle X-ray scattering (SAXS) and compared the
enzyme structure with the known crystal structure of the monomeric PEPCK from Escherichia coli. The dimeric structure possesses 2-fold symmetry, with each monomer sharing a high degree of structural similarity with the monomeric structure of the E. coli PEPCK. Each monomer folds into two complex mixed alpha/beta domains, with the active site located in a deep cleft between the domains. The two active sites in the dimer are far apart from each other, in an arrangement that seems to permit an independent access of the substrates to the two active sites. All residues of the E. coli PEPCK structure that had been found to interact with substrates and
metal cofactors have been found conserved and in a substantially equivalent spatial disposition in the T. cruzi PEPCK structure. No substrate or
metal ion was present in the crystal structure. A sulphate ion from the crystallisation medium has been found bound to the active site.
Solution SAXS data suggest that, in solutions with lower sulphate concentration than that used for the crystallisation experiments, the actual
enzyme conformation may be slightly different from its conformation in the crystal structure. This could be due to a conformational transition upon sulphate binding, similar to the
ATP-induced transition observed in the E. coli PEPCK, or to crystal packing effects. The present structure of the T. cruzi PEPCK will provide a good basis for the modelling of new anti-chagasic
drug leads.