Allosteric Regulation of Trypanosoma bruceiRibonucleotide Reductase Studied in Vitro and in Vivo

Abstract

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 dNTPs needed for DNA synthesis. Results with the isolated recombinant trypanosome ribonucleotide reductase showed that dATP specifically directs pyrimidine ribonucleotide reduction instead of being a general negative effector as in other related ribonucleotide reductases, whereas dTTP and dGTP directed GDP and ADP reduction, respectively. Pool measurements of NDPs, NTPs, and dNTPs in the cultivated bloodstream form of trypanosomes exposed to deoxyribonucleosides or inhibited by hydroxyurea confirmed our in vitro allosteric regulation model of ribonucleotide reductase. Interestingly, the trypanosomes had extremely low CDP and CTP pools, whereas the dCTP pool was comparable with that of other dNTPs. The trypanosome ribonucleotide reductase seems adapted to this situation by having a high affinity for the CDP/UDP-specific effector dATP and a high catalytic efficiency, Kcat/Km, for CDP reduction. 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 proliferation, a valuable observation in the search for new trypanocidal nucleoside analogues.