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

Abstract

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