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20102
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Subject: Isoenzymes.

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Now showing 1 - 2 of 2
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    Influence of Active Site Ligands and Nanoparticle Surfaces on Human Carbonic Anhydrase Isozymes
    (North Dakota State University, 2010) Manokaran, Sumathra
    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.
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    Differential Modulation of the Structural and Functional Characteristics of Human Matrix Metalloproteinase Isozymes upon Binding to Different Ligands
    (North Dakota State University, 2010) Ganguly, Bratati
    Matrix metalloproteinases (MMPs) are a family of Zn2 + -dependent, Ca2 + -containing endoproteinases involved in tissue remodeling and degradation of the extracellular matrix (ECM). Human MMP isozymes are known to be involved in the progression and metastasis of many diseases like cancer, Alzheimer's, and etc. The different nanoparticles (e.g. gold nanoparticles, liposomes, and charged quantum dots) used in this study provides insights into nanoparticle-induced differential modulation in the structural-functional characteristics of MMP 7, 9 and 10 for better therapeutic intervention. To demonstrate the relationship between the rigid and flexible surfaces on the differential modulation of functional and structural characteristics of MMP-7, polylysine (PLL) and cationic gold nanoparticles (Au-CNP) were selected as representative examples. These cationic nano-structures were expected to serve as "soft" (flexible) and ''rigid" (hard) ligands, respectively. Steady-state kinetic analysis demonstrated that PLL induces activation and inhibition of MMP-7 at stoichiometric and super-stoichiometric concentrations respectively. Circular Dichroism spectroscopy was used to confirm that binding of Au-CNP to MMP-7 induces denaturation of the protein. In pursuit of understanding the molecular origin of the intrinsic selectivity in binding of human MMP isozymes to differently charged lipid membranes, steady-state kinetic studies and intrinsic tryptophan quenching studies were carried out. Results demonstrated that differently charged lipid membranes bind to all three MMPs; phosphotidylserine (POPS) liposomes are selective for MMP-7. The bipolar distribution of negative and positive charges on the surface of this enzyme dictates the binding of liposomes and perturbation of catalytic activity. An attempt to explain the molecular rationale for alternative binding modes of differently charged quantum dots (QDs) to the three MMPs, steady-state tryptophan quenching, steady-state kinetics, and time-resolved fluorescence measurements were carried out. Differently charged QDs bind to all the three MMP isozymes. Enzyme activity of these MMPs was perturbed upon binding to cationic and anionic QDs. Binding of MMPs to the differently charged QDs is reversible and is mediated via electrostatic interactions. Analysis of time-resolved fluorescence data indicates that the protein expenences different micro-environments, due to different distribution of intrinsic tryptophan residues (buried and exposed) on MMP isozymes or the existence of two distinct conformations of the protein. Binding to charged QDs perturbs enzyme activity of MMPs either by restricting the access of the substrate to the active-site or through allosteric modulation. In order to develop new isozyme-selective inhibitors, small molecule inhibitors (SMis) were designed, synthesized and screened for MMP-7, 9 and 10. Results indicate that hydroxamates and carboxylates are preferred SMis. Binding preference is based on either the micro-environments of the active-site pockets.