Thermodynamic characterization of substrate and inhibitor binding to Trypanosoma brucei 6-phosphogluconate dehydrogenase.
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.
Montin
Katy
K
Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, Italy.
Cervellati
Carlo
C
Dallocchio
Franco
F
Hanau
Stefania
S
D000818:Animals; D002151:Calorimetry / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005942:Gluconates / Q000737:chemistry / Q000378:metabolism; D006877:Hydroxamic Acids / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010734:Phosphogluconate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013400:Sugar Acids / Q000737:chemistry / Q000494:pharmacology; D013403:Sugar Phosphates / Q000737:chemistry / Q000494:pharmacology; D013816:Thermodynamics; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
C020462:4-phosphoerythronate; C518606:4-phosphoerythronohydroxamic acid; D004791:Enzyme Inhibitors; D005942:Gluconates; D006877:Hydroxamic Acids; D015800:Protozoan Proteins; D013400:Sugar Acids; D013403:Sugar Phosphates; D009249:NADP; D010734:Phosphogluconate Dehydrogenase; C008884:6-phosphogluconic acid