6-Phosphogluconate dehydrogenase is a potential target for new drugs against
African trypanosomiasis. Phosphorylated aldonic
acids are strong inhibitors of
6-phosphogluconate dehydrogenase, and 4-phospho-d-erythronate (4PE) and 4-phospho-d-erythronohydroxamate are two of the strongest inhibitors of the Trypanosoma brucei
enzyme. Binding of the substrate 6-phospho-d-gluconate (6PG), the inhibitors 5-phospho-d-ribonate (5PR) and 4PE, and the
coenzymes NADP,
NADPH and
NADP analogue 3-amino-pyridine
adenine dinucleotide
phosphate to 6-phospho-d-gluconate
dehydrogenase from T. brucei was studied using isothermal titration calorimetry. Binding of the substrate (K(d) = 5 microm) and its analogues (K(d) =1.3 microm and K(d) = 2.8 microm for 5PR and 4PE, respectively) is entropy driven, whereas binding of the
coenzymes is enthalpy driven. Oxidized
coenzyme and its analogue, but not reduced
coenzyme, display a half-site reactivity in the ternary complex with the substrate or inhibitors. Binding of 6PG and 5PR poorly affects the dissociation constant of the
coenzymes, whereas binding of 4PE decreases the dissociation constant of the
coenzymes by two orders of magnitude. In a similar manner, the K(d) value of 4PE decreases by two orders of magnitude in the presence of the
coenzymes. The results suggest that 5PR acts as a substrate analogue, whereas 4PE mimics the transition state of dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the mechanism of the
enzyme, suggesting that the inhibitor forces the catalytic
lysine 185 into the protonated state.