| dc.description.abstract | Carbonic anhydrase (CA) is an ubiquitously distributed zinc containing metallo
enzyme that catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a
proton. Existence of 16 isoenzymes of CA in the animal kingdom has been known so far
with varying subcellular and tissue distributions. Due to their involvement in many
physiological and pathological processes, these isozymes have been the target for drug
designing for the past 6 decades. The present study was designed with the aim of
understanding the effect of active site ligands and nanoparticle surfaces on human carbonic
anhydrase isozymes.
In an effort to identify a fluorescent probe for carbonic anhydrases, the quantum
yields and binding affinities of a variety of naphthalenesulfonamide derivatives with
human carbonic anhydrase isozymes (hCAs) were determined. In this pursuit, a highly
sensitive fluorescent probe, JB2-48 was identified. Experiments involving the above
flurophore with hCA I unraveled the contributions of the sulfonamide moiety and the
hydrophobic regions of the ligand structure on the spectral, kinetic, and thermodynamic
properties of the enzyme-ligand complex. The fluorescence 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 ligand. This is manifested via
the electrostatic interaction between the active site resident Zn2
+ cofactor and the
negatively charged sulfonamide group of the ligand. Evidence was provided that the anionic and neutral forms of JB2-48 are stabilized by the complementary
microscopic/conformational states of the enzyme.
Investigations on the binding of the sulfonamide inhibitor, benzene sulfonamide
(BS), with hCA isozymes II and VII, revealed that the binding is stabilized by polar
interactions in the former case and hydrophobic interactions in the latter case. In addition, it
was found that the binding of BS with hCA II is enthalpically driven at low temperatures,
whereas it is entropically driven for its binding with hCA VII.
Due to the prevalence of bipolar distribution of charges on hCA XII, the effects of
the interaction of differently charged quantum dots, liposomes and polylysine on hCA XII
were investigated. These charged particles were found to differently modulate the active
site of the enzyme. The data revealed that whereas poly lysine and liposomes exhibited no
influence on the binding and catalytic features of the enzyme, quantum dots had significant
influence on the above features. Arguments were presented that the above differential
feature exhibited by quantum dots, liposomes and poly lysine is encoded in the rigidity
versus flexibility of the charged molecules.
Studies on the denaturation of hCA isozymes II and XII unraveled their unfolding
mechanism. It was found that the unfolding ofhCA XII followed a simple two state model
from native to unfolded state; however hCA II unfolded with the formation of a stable
intermediate. | en_US |
