Allosteric regulation of Trypanosoma brucei ribonucleotide reductase studied in vitro and in vivo

Trypanosoma brucei is the causative agent for African sleeping sickness, We have made in vitro and in vivo studies on the allosteric regulation of the trypanosome ribonucleotide reductase, a key enzyme in the production of dN TPs needed for DNA synthesis. Results with the isolated recombinant trypano some ribonucleotide reductase showed that dATP specifically directs pyrimid ine ribonucleotide reduction instead of being a general negative effector a s in other related ribonucleotide reductases, whereas dTTP and dGTP directe d GDP and ADP reduction, respectively. Pool measurements of NDPs, NTPs, and dNTPs in the cultivated bloodstream form of trypanosomes exposed to deoxyr ibonucleosides or inhibited by hydroxyurea confirmed our in vitro allosteri c regulation model of ribonucleotide reductase, Interestingly, the trypanos omes had extremely low CDP and CTP pools, whereas the dCTP pool was compara ble with that of other dNTPs, The trypanosome ribonucleotide reductase seem s adapted to this situation by having a high affinity for the CDP/UDP-speci fic effector dATP and a high catalytic efficiency, K-cat/K-m, for CDP reduc tion. Thymidine and deoxyadenosine were readily taken up and phosphorylated to dTTP and dATP, respectively, the latter in a nonsaturating manner. This uncontrolled uptake of deoxyadenosine strongly inhibited trypanosome proli feration, a valuable observation in the search for new trypanocidal nucleos ide analogues.