Protonation enthalpies for the active site residues of Trypanosoma brucei 6-phosphogluconate dehydrogenase

Inversions of “natural” ionization state of the ionisable residues in the active sites are frequently reported, but rarely it is known their energy cost. Here we report the energy cost of setting in the correct protonation state the active site of 6-phosphogluconate dehydrogenase (6PGDH) from T. brucei. 6PGDH is the third enzyme of the pentose phosphate pathway, catalyzing the NADP-dependent oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose-5-phosphate (RU5P). 6PG binding to 6PGDH has been studied by ITC in the WT and mutants in the two residues mainly involved in catalysis, K185 and E192, whose protonation state invert upon the binding. From the pH dependence of the binding and activity it can be seen that binding of 6PG raises the pKa of E192 from 7.07 to 9.64, while it decreases the pKa of K185 from 9.9 to 7.17. E192 protonation gives a favourable enthalpy of ≈ -1.5 kcal/mole, while Lys185 deprotonation has an unfavourable enthalpy of ≈ +7.6 kcal/mole, with a net energy cost for the ionization exchange of 6.1 kcal/mole at the expense of the binding enthalpy. Since the ionization state of K185 and E192 in the enzyme-product complex is the same as in the free enzyme, thus the energy spent during 6PG binding will be used in driving the catalytic cycle. It is noteworthy that a theoretical model of 6PGDH catalysis predict that the non protonated K185 decreases the energy of the transition state of the dehydrogenation reaction by about 7.0 kcal/mole (1). (1) Wang J and Li S. Catalytic Mechanism of 6-Phosphogluconate Dehydrogenase: A Theoretical Investigation J. Phys. Chem. B 2006, 110, 7029-703

NADPH reduces oligomerization rate of a pre-existing dimer-tetramer equilibrium inTrypanosoma brucei 6-phosphogluconate dehydrogenase

6--Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the NADP-dependent oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose-5-phosphate (RU5P). It not only gives NADPH and RU5P, but also depletes 6PG, whose accumulation induces cell senescence. It is a proposed drug target for African trypanosomiasis caused by T. brucei and for other microbial infections and cancer. We report here that the association of dimers to tetramers is an equilibrium present in the free enzyme, independently of the ligands. It has been shown by glutaraldehyde cross-linking, dynamic light scattering (DLS) and density gradient sedimentation. Both DLS and sedimentation indicate the enzyme size increases by increasing the enzyme concentration. In addition, gel filtration, density gradient sedimentation and isothermal titration calorimetry (ITC) reveal that the oligomerization rates are differently influenced by ligands. Indeed dynamic experiments where different oligomeric forms can be separated, show a strong NADPH shift of the enzyme versus the tetrameric form while NADP does not affect dimeric state of 6PGDH. Accordingly heat capacity change measured by ITC is different between NADP and NADPH binding (-92.56 against -520.35 cal/mol∙K) in agreement with a decreased solvent exposed surface area in tetramer. Tetramer is about 3-fold more active than dimer, indeed NADPH, the 6PGDH product and inhibitor, decreases interconversion rate while 6PG antagonizes NADPH effect. This is a further substrate way of promoting increased catalytic efficiency. The sheep liver 6PGDH, instead, by sedimentation studies appears always a dimer, the dimertetramer shift hence representing further drug exploitable potential

Protonation enthalpies for the active site residues of Trypanosoma brucei 6-phosphogluconate dehydrogenase

Inversions of “natural” ionization state of the ionisable residues in the active sites are frequently reported, but rarely it is known their energy cost. Here we report the energy cost of setting in the correct protonation state the active site of 6-phosphogluconate dehydrogenase (6PGDH) from T. brucei. 6PGDH is the third enzyme of the pentose phosphate pathway, catalyzing the NADP-dependent oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose-5-phosphate (RU5P). 6PG binding to 6PGDH has been studied by ITC in the WT and mutants in the two residues mainly involved in catalysis, K185 and E192, whose protonation state invert upon the binding. From the pH dependence of the binding and activity it can be seen that binding of 6PG raises the pKa of E192 from 7.07 to 9.64, while it decreases the pKa of K185 from 9.9 to 7.17. E192 protonation gives a favourable enthalpy of ≈ -1.5 kcal/mole, while Lys185 deprotonation has an unfavourable enthalpy of ≈ +7.6 kcal/mole, with a net energy cost for the ionization exchange of 6.1 kcal/mole at the expense of the binding enthalpy. Since the ionization state of K185 and E192 in the enzyme-product complex is the same as in the free enzyme, thus the energy spent during 6PG binding will be used in driving the catalytic cycle. It is noteworthy that a theoretical model of 6PGDH catalysis predict that the non protonated K185 decreases the energy of the transition state of the dehydrogenation reaction by about 7.0 kcal/mole (1). (1) Wang J and Li S. Catalytic Mechanism of 6-Phosphogluconate Dehydrogenase: A Theoretical Investigation J. Phys. Chem. B 2006, 110, 7029-703

Evidence for dimer/tetramer equilibrium in Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer-tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in Cp, with NADP binding Cp closer to what is expected of crystallographic structures, while NADPHCp is three times larger. The estimated dimer-tetramer association constant is 1.5 106 M-1, and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme

Energy cost for the proper ionization of active site residues in 6-phosphogluconate dehydrogenase from T. brucei

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 mutants enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈ 6.1 kcal/mole. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240-325 cal/mol. However proton balance evidences the dissociation of other(s) 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 1.2-2.3 kcal/mol to set the enzyme into a catalytically competent form

Finding peculiar patterns of kinetoplastida enzymes to be exploited in drug design

If a parasite and host metabolic ways differ, such as an enzyme is present only in parasite, obviously this is a good target in drug design. Also, essential enzymes present in both can be good targets if we exploit selective patterns of the parasite enzyme. At this aim, comparison of the pentose phosphate pathway (PPP) 2nd dehydrogenase, 6-phosphogluconate dehydrogenase (6PGDH), between mammal host and Trypanosoma brucei, has been done in our lab. PPP provides above all necessary NADPH to all cell reduction reactions, including those contrasting both oxidative, parasite environment and host response. Although kinetoplastids 6PGDH shows only 33% amino acid identity with mammal 6PGDH, 3D-structure and general acid-base mechanism are similar, with many conserved residues in the active site. Anyway, by studying enzymes in more detail, several differences were found. Looking for the affinity of some polycyclic compounds, preferential binding of T. brucei 6PGDH to triphenylmethane with either nitrogen or oxygen as substituent in two rings and a sulfonate in the 3rd ring was found compared to the mammal enzyme. A selectivity of 40 was shown by Brilliant Green and this is probably due to the shorter distance between two active site lysines than in mammal. By studying homotropic cooperativity other significant differences were found. In fact substrate binding to one subunit increases homodimer catalytic efficiency; even if the two subunits have an identical sequence they are implied in different steps. This was shown by several ways, for instance in presence of the substrate 6PG there is a NADP half-site reactivity, also 6PG activates decarboxylation following 6PG oxidation, but differently from the liver enzyme the parasite's one is able to catalyse this step even in absence of an activator. Besides, allosteric modulation for the parasite enzyme is not apparent when reverse reaction is studied, while it is shown by yeast enzyme, which is very similar to the liver's one. Thus, diversity exists which allowed to find ligands more than 250-fold selective, as transition state analogues 4-phospho-erythronoxamate and 4-phospho-erythronate. This characterization has disclosed lead compounds for medicinal chemists to transform those into pro-drugs and derivatives with good stability and solubility. Last difference found is that parasite 6PGDH shows a ligand-modulated tetramerization, which is not present in mammal enzyme representing further drug exploitable potential