Stabilization of anionic and neutral forms of a fluorophoric ligand at the active site of human carbonic anhydrase I

作者：Manokaran Sumathra; Banerjee Jayati; Mallik Sanku; Srivastava D K^*

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2010, 1804(10): 1965-1973.

DOI：10.1016/j.bbapap.2010.06.024

• Human carbonic anhydrase-I
• Inhibitor design
• Sulfonamide
• Fluorophore
• Dansylamide
• Ligand binding

We synthesized a fluorogenic dansylamide derivative (JB2-48), which fills the entire (15 angstrom deep) active site pocket of human carbonic anhydrase I, and investigated the contributions of sulfonamide and hydrophobic regions of the ligand structure on the spectral, kinetic, and thermodynamic properties of the enzyme-ligand complex. The steady-state and fluorescence lifetime data revealed that the deprotonation of the sulfonamide moiety of the enzyme bound ligand increases the fluorescence emission intensity as well as the lifetime of the fluorophores. This is manifested via the electrostatic interaction between the active site resident Zn(2+) cofactor and the negatively charged sulfonamide group of the ligand, and such interaction contributes to about 2.2 kcal/mol (Delta Delta G degrees) and 0.89 kcal/mol (Delta Delta G dagger) energy in stabilizing the ground and the putative transition states, respectively. We provide evidence that the anionic and neutral forms of JB2-48 are stabilized by the complementary microscopic/conformational states of the enzyme. The implication of the mechanistic studies presented herein in rationale design of carbonic anhydrase inhibitors is discussed.

• 出版日期2010-10