Premium
Intrinsic thermodynamics of trifluoromethanesulfonamide and ethoxzolamide binding to human carbonic anhydrase VII
Author(s) -
Pilipuitytė Vilma,
Matulis Daumantas
Publication year - 2015
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.2404
Subject(s) - chemistry , isothermal titration calorimetry , carbonic anhydrase , gibbs free energy , carbonic anhydrase ii , deprotonation , protonation , enthalpy , allosteric regulation , stereochemistry , biochemistry , thermodynamics , enzyme , organic chemistry , ion , physics
Human carbonic anhydrase (CA) isozyme VII is a cytosolic protein that is highly expressed in the cortex, hippocampus, and thalamus regions within mammalian brain, and expression disorders can cause epilepsy and several cases of malignant brain tumors. Therefore, CA VII is a potential antiepileptic and anticancer drug target. There are numerous sulfonamides that target CAs nonspecifically. It is important to understand the thermodynamics of inhibitor binding and the structural features of the protein–inhibitor complex in order to design specific inhibitors against CA VII. Isothermal titration calorimetry and fluorescent thermal shift assay were used to characterize the intrinsic thermodynamic parameters of trifluoromethanesulfonamide and ethoxzolamide binding to CA VII. Binding experiments were carried out at various pH in different buffers in order to dissect linked protonation of the water molecule bound to the CA VII active site, deprotonation of the sulfonamide group of the inhibitor, and protonation–deprotonation of buffer. Dissection of all those contributions yielded the intrinsic thermodynamic parameters of binding, such as Gibbs free energy, binding enthalpy, entropy, and protein p K a value. Thermal shift assay was also used to determine CA VII stability at various pH. Copyright © 2015 John Wiley & Sons, Ltd.