Thermodynamic characterization of substrate and inhibitor binding to Trypanosoma brucei 6‐phosphogluconate dehydrogenase

Abstract

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 (Kd = 5 µm) and its analogues (Kd =1.3 µm and Kd = 2.8 µm 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 Kd 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.

6-Phosphogluconate Dehydrogenase Mechanism

“…These data indicate that after the dehydrogenation step, the enzyme works more efficiently when one subunit is still occupied by the substrate. This could correlate with the half-site reactivity observed for the coenzyme binding in the ternary complex enzyme-6PG-NADP (10,20,21). In fact, if only one NADP is bound to the dimeric enzyme in the ternary complex, a second 6PG could remain unchanged on the second subunit to assist the subsequent reaction steps.…”

Conformational changes associated with cofactor/substrate binding of 6-phosphogluconate dehydrogenase from Escherichia coli and Klebsiella pneumoniae: Implications for enzyme mechanism

“…Moreover, our structures also provide information for antibiotics development. Indeed, some inhibitors of Tb6PGDH have been prepared (Montin et al, 2007;Ruda et al, 2007).…”

“…7A). Each enzyme subunit first interacts with 6PG leading to the conformational rearrangement of the enzyme (Montin et al, 2007) and leaves one of subunits with an ''open" conformation, anchoring a coenzyme molecule by hydrogen bonding with the adenosine 2 0 -phosphate. On the other hand, N102 and M14 hold the adenosine ribose and assist the formation of the closed structure.…”

“…It is noteworthy that none of the triad residues is bound to the Nam ring of Nbr 8 ADP in Oa6PGDH/Nbr 8 ADP binary complex and these different binding modes of the oxidized and reduced cofactors infer that NADPH is involved in the orientation of substrate and facilitating decarboxylation 3-keto 6PG (Cervellati et al, 2008;. In addition, structural and thermodynamic studies suggested a modulation of the conformational change for a single subunit of the homodimer preceding catalysis (Montin et al, 2007;Sundaramoorthy et al, 2007). When it complexed with the product or inhibitors, a difference of cofactor binding in the coenzymebinding domain of the two subunits in the homodimeric 6PGDH was observed (Sundaramoorthy et al, 2007), which suggested the half-of-the-sites enzyme mechanism (Montin et al, 2007).…”

“…In addition, structural and thermodynamic studies suggested a modulation of the conformational change for a single subunit of the homodimer preceding catalysis (Montin et al, 2007;Sundaramoorthy et al, 2007). When it complexed with the product or inhibitors, a difference of cofactor binding in the coenzymebinding domain of the two subunits in the homodimeric 6PGDH was observed (Sundaramoorthy et al, 2007), which suggested the half-of-the-sites enzyme mechanism (Montin et al, 2007). However, until recently, it is unclear why occupancy of one active site has an influence on the other.…”