title: Characterization of human carbonic anhydrase XII stability and inhibitor binding.
abstract: Human carbonic anhydrase isozyme XII is a transmembrane protein that is overexpressed in many human cancers. Therefore CA XII is an anticancer drug target. However, there are few compounds that specifically target CA XII. The design of specific inhibitors against CA XII relies on the detailed understanding of the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex. To characterize the thermodynamic parameters of the binding of known sulfonamides, namely ethoxzolamide, acetazolamide and trifluoromethanesulfonamide, we used isothermal titration calorimetry and fluorescent thermal shift assay. The binding of these sulfonamides to CA XII was buffer and pH-dependent. Dissection of protonation-deprotonation reactions of both the water molecule bound to the CA XII active site and the sulfonamide group of the inhibitor yielded the intrinsic thermodynamic parameters of binding, such as binding enthalpy, entropy and Gibbs free energy. Thermal shift assay was also used to determine CA XII stabilities at various pH and in the presence of buffers and salts.
authors.affiliation: Department of Biothermodynamics and Drug Design, Vilnius University Institute of Biotechnology, Graičiūno 8, Vilnius LT-02241, Lithuania.
authors.forename: Vaida
authors.initials: V
authors.lastname: Jogaitė
authors.forename: Asta
authors.initials: A
authors.lastname: Zubrienė
authors.forename: Vilma
authors.initials: V
authors.lastname: Michailovienė
authors.forename: Joana
authors.initials: J
authors.lastname: Gylytė
authors.forename: Vaida
authors.initials: V
authors.lastname: Morkūnaitė
authors.forename: Daumantas
authors.initials: D
authors.lastname: Matulis
mesh_terms: D002151:Calorimetry; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D055550:Protein Stability; D012492:Salts / Q000737:chemistry; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D012492:Salts; D013449:Sulfonamides; D002256:Carbonic Anhydrases; C477378:carbonic anhydrase XII