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The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pKa of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pKa 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pKa, 7.17 and 9.64, in the enzyme-substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pKa from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pKa from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈6.1kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240-325cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pKa shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2-2.3kcal/mol to set the enzyme into a catalytically competent form., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG (D)(V/K) and (D)(V) are 1.68 and 2.46, respectively, whereas the presence of 6PG increases (D)(V/K) to 2.84 and decreases (D)(V) to 1.38. A similar increase of (T)(V/K) is observed as 6PG builds up in the reaction mixture. These data indicate that in the absence of 6PG, a slow step, which precedes the chemical process, is rate-limiting for the reaction, whereas in the presence of 6PG, the rate-limiting step follows the isotope-sensitive step. Kinetic analysis of reductive carboxylation shows that 6PG at low concentrations decreases the K(m) of Ru5P, whereas at higher concentrations, the usual competitive pattern is observed. These data indicate that full activity of 6PGDH is achieved when one subunit carries out the catalysis and the other subunit carries an unreacted 6PG. Thus, 6PG is like an allosteric activator of 6PGDH.

A recent study suggested sheep liver 6-phosphogluconate dehydrogenase (6PGDH) sees the oxidized and reduced cofactor differently [Cervellati, C., Dallocchio, F., Bergamini, C. M., and Cook, P. F. (2005) Biochemistry 44, 2432-2440]. Data were consistent with a rotation into the active site of the nicotinamide ring of NADP upon its reduction, resulting in a displacement of the 1-carboxylate of 3-keto-6PG better positioning it for decarboxylation, and further suggested a role of the cofactor in generating the precatalytic conformation of the enzyme. To further probe the role of the cofactor, multiple isotope effects were measured for the enzyme with mutations of the two residues that directly interact with the nicotinamide ring of NADP+, methionine 13 and glutamate 131. Kinetic and isotope effect data obtained in this study will thus be interpreted in terms of a mechanism that includes the rotation of the nicotinamide ring. The M13V, M13Q, M13C, and E131A mutant enzymes were characterized with respect to their kinetic parameters, deuterium, 13C, multiple deuterium/13C isotope effects, and the kinetics of utilization of 2-deoxy-6PG. Data suggest the position of the nicotinamide ring is important in identifying the open and closed conformations of the enzyme, with the latter optimal for catalysis. The 6PGDH reaction provides an excellent example of the use of substrate binding energy to drive catalysis.

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.

The crystal structure of sheep liver 6-phosphogluconate dehydrogenase (6PGDH) shows marked differences in the position of the nicotinamide mononucleotide (NMN) moiety of NADP(+) and NADPH (Adams, J. M., Grant, H. E., Gover, S., Naylor, C. E., and Phillips, C. (1994) Structure 2, 651-668). A methionine side chain (Met13) interacts with the si face of NADP(+) in the complex with the oxidized coenzyme, is likely to affect the binding mode of the nicotinamide ring of NADP(+), and may play a role in catalysis in the 6PGDH reaction. To check this possibility we performed site-directed mutagenesis, changing M13 to a number of residues including V, I, C, F, and Q. Mutant enzymes were characterized with respect to their kinetic parameters and primary deuterium isotope effects. All mutations resulted in a decrease in affinity of the enzyme for NADP(+), but not NADPH. In addition, the M13 to C (M13C), M13F, and M13Q mutant enzymes exhibited a decrease of at least an order of magnitude in V/E(t). The deuterium isotope effects on V and V/K(6PG) were decreased to about 1.2 for the M13F and M13C mutant enzymes, while they were increased to about 2.4 for the M13Q enzyme (a value of 1.8-1.9 is obtained for the wild-type enzyme). In at least three instances changes in the overall rate of the oxidative decarboxylation reaction relative to other steps along the reaction pathway were observed. Isotope effects indicate that the hydride transfer steps can become either more or less rate-determining dependent on the substitution. Data are consistent with a significant role of M13 in the orientation of the cofactor nicotinamide ring in the mechanism of 6PGDH, likely with respect to geometry and distance of the ring from C3 of 6PG.

Sugar derivatives mimicking compounds which take part in the catalysed reaction have been assayed as alternative substrates and/or competitive inhibitors of 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Phosphonate analogues have been synthesised and the new compound 5-deoxy-5-phosphono-D-arabinonate shows good selectivity towards the parasite enzyme. A number of 4-carbon and 5-carbon aldonates are strong inhibitors of the parasite enzyme with K(i) values below the substrate K(m) and some acyl derivatives are also potent inhibitors. At least five of the compounds showing a significant selectivity for the parasite enzyme represent leads for trypanocidal drugs against this recently validated target.