Inhibitor binding to metal-substituted metalloenzyme: Sulfonamide affinity for carbonic anhydrase IX

• Published in: Journal of inorganic biochemistry 2024-07, Vol.256, p.112547, Article 112547
• Main Authors: Baronas, Denis, Knašienė, Birutė, Mickevičiūtė, Aurelija, Jachno, Jelena, Naujalis, Evaldas, Zubrienė, Asta, Matulis, Daumantas
• Format: Article
• Language: English
• Subjects: Divalent metal ion; Fluorescent thermal shift assay; Metal-free carbonic anhydrase (apoCA); Metal-substituted carbonic anhydrase; Metalloenzyme; Sulfonamide
• ISSN: 0162-0134; 1873-3344; 1873-3344
• DOI: 10.1016/j.jinorgbio.2024.112547

Transition metal ions are structural and catalytic cofactors of many proteins including human carbonic anhydrase (CA), a Zn-dependent hydrolase. Sulfonamide inhibitors of CA recognize and form a coordination bond with the Zn ion located in the active site of the enzyme. The Zn ion may be removed or substituted with other metal ions. Such CA protein retains the structure and could serve as a tool to study metal ion role in the recognition and binding affinity of inhibitor molecules. We measured the affinities of selected divalent transition metal ions, including Mn, Fe, Co, Ni, Cu, Cd, Hg, and Zn to metal-free CA isozymes CA I, CA II, and CAIX by fluorescence-based thermal shift assay, prepared metal-substituted CAs, and determined binding of diverse sulfonamide compounds. Sulfonamide inhibitor binding to metal substituted CA followed a U-shape pH dependence. The binding was dissected to contributing binding-linked reactions and the intrinsic binding reaction affinity was calculated. This value is independent of pH and protonation reactions that occur simultaneously upon binding native CA and as demonstrated here, to metal substituted CA. Sulfonamide inhibitor binding to cancer-associated isozyme CAIX diminished in the order: Zn > Co > Hg > Cu > Cd > Mn > Ni. Energetic contribution of the inhibitor-metal coordination bond was determined for all above metals. The understanding of the principles of metal influence on ligand affinity and selectivity should help design new drugs targeting metalloenzymes. Zinc was removed from the active site of carbonic anhydrase CAIX and affinities of divalent metals determined. Inhibitor binding to metal-substituted CA was dependent on pH and the binding was linked to protonation reactions that need to be subtracted to obtain the intrinsic affinity of inhibitor binding to metal-substituted CA. [Display omitted] •Ligand affinities for carbonic anhydrases by thermal shift assay spanned a large dynamic range from millimolar to picomolar.•Metal-substituted carbonic anhydrases follow the rule of sulfonamide intrinsic binding energetics.•Compound binding to metal-substituted carbonic anhydrases showed energetic additivity for drug design.