title: Energy cost for the proper ionization of active site residues in 6-phosphogluconate dehydrogenase from T. brucei.
abstract: 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.
authors.affiliation: Department of Biomedical and Specialty Surgical Sciences, Biochemistry Unit, Via Borsari 46, Università di Ferrara, 44121 Ferrara, Italy. Electronic address: hns@unife.it.
authors.forename: S
authors.initials: S
authors.lastname: Hanau
authors.affiliation: Department of Biomedical and Specialty Surgical Sciences, Biochemistry Unit, Via Borsari 46, Università di Ferrara, 44121 Ferrara, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Proietti d'Empaire
authors.affiliation: Department of Biomedical and Specialty Surgical Sciences, Biochemistry Unit, Via Borsari 46, Università di Ferrara, 44121 Ferrara, Italy.
authors.forename: K
authors.initials: K
authors.lastname: Montin
authors.affiliation: Department of Biomedical and Specialty Surgical Sciences, Biochemistry Unit, Via Borsari 46, Università di Ferrara, 44121 Ferrara, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Cervellati
authors.affiliation: Department of Biomedical and Specialty Surgical Sciences, Biochemistry Unit, Via Borsari 46, Università di Ferrara, 44121 Ferrara, Italy.
authors.forename: I
authors.initials: I
authors.lastname: Capone
authors.affiliation: Department of Biomedical and Specialty Surgical Sciences, Biochemistry Unit, Via Borsari 46, Università di Ferrara, 44121 Ferrara, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Dallocchio
mesh_terms: D020134:Catalytic Domain; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010734:Phosphogluconate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011522:Protons; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics; D014346:Trypanosoma brucei brucei / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics
chemical_list: D011522:Protons; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010734:Phosphogluconate Dehydrogenase; D008239:Lysine
keywords: 6-Phosphogluconate dehydrogenase; Active site pK(a); ITC; Ionization enthalpy; Proton linkage; Trypanosoma brucei