title: Alternating sites reactivity is a common feature of thiamin diphosphate-dependent enzymes as evidenced by isothermal titration calorimetry studies of substrate binding.
abstract: Thiamin diphosphate (ThDP)-dependent enzymes play vital roles in cellular metabolism in all kingdoms of life. In previous kinetic and structural studies, a communication between the active centers in terms of a negative cooperativity had been suggested for some but not all ThDP enzymes, which typically operate as functional dimers. To further underline this hypothesis and to test its universality, we investigated the binding of substrate analogue methyl acetylphosphonate (MAP) to three different ThDP-dependent enzymes acting on substrate pyruvate, namely, the Escherichia coli E1 component of the pyruvate dehydrogenase complex, E. coli acetohydroxyacid synthase isoenzyme I, and the Lactobacillus plantarum pyruvate oxidase using isothermal titration calorimetry. The results unambiguously show for all three enzymes studied that only one active center of the functional dimers accomplishes covalent binding of the substrate analogue, supporting the proposed alternating sites reactivity as a common feature of all ThDP enzymes and resolving the recent controversy in the field.
authors.affiliation: Göttingen Center for Molecular Biosciences, Georg-August-University Göttingen, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11,37077 Göttingen, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Schröder-Tittmann
authors.forename: Danilo
authors.initials: D
authors.lastname: Meyer
authors.forename: Johannes
authors.initials: J
authors.lastname: Arens
authors.forename: Cindy
authors.initials: C
authors.lastname: Wechsler
authors.forename: Michael
authors.initials: M
authors.lastname: Tietzel
authors.forename: Ralph
authors.initials: R
authors.lastname: Golbik
authors.forename: Kai
authors.initials: K
authors.lastname: Tittmann
mesh_terms: D000095:Acetolactate Synthase / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D020134:Catalytic Domain; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000737:chemistry / Q000378:metabolism; D011771:Pyruvate Oxidase / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D013835:Thiamine Pyrophosphate / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004798:Enzymes; D029968:Escherichia coli Proteins; C014660:methylacetylphosphonate; D011771:Pyruvate Oxidase; D030481:Pyruvate Dehydrogenase (Lipoamide); D000095:Acetolactate Synthase; D010746:Phosphonoacetic Acid; D013835:Thiamine Pyrophosphate