Truppo, Emanuela (2011) BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF HUMAN CARBONIC ANHYDRASES. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||carbonic anhydrase; enzyme inhibition; structure-based drug design.|
|Date Deposited:||07 Dec 2011 09:02|
|Last Modified:||17 Jun 2014 06:04|
Carbonic anhydrases (CAs. EC 188.8.131.52) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and proton. In humans, 15 isozymes have been described with different subcellular localization. Indeed, CA I-III, VII, and XIII are cytosolic, CA IV, IX, XII and XIV are membrane-bound, CA VA and CA VB are mitochondrials, and CA VI is secreted. Human CA isozymes are extensively distributed in several tissues and organs where, as modulators of pH and ion transport, they take part in a variety of physiological and pathological processes. As a result, in the last years many of these enzymes have become important therapeutic targets for pharmaceutical research. However, given the high degree of sequence and structure similarity among the different isoforms, to date most of the CA-directed drugs developed lack of selectivity and consequently present many side-effects. X-Ray crystallography is one of the most useful instruments in the structure-based drug design of selective molecules able to interact with a target enzyme. Indeed, there are several examples in the literature where knowledge of the crystallographic structure of an enzyme allowed the design of molecules able to interact with specific residues, thus regulating enzyme biological activity. This is why in recent years there has been an extensive research effort focusing on the crystal structure resolution of all catalytically active alfa-CA isoforms, with the result that most of the human CA isoforms have been so far structurally characterized. As a part of a general research project based on the structure-based drug design of isoform-selective CA inhibitors (CAIs), my Ph.D thesis has been focused on the study of the biochemical and structural features of two cytosolic CA isoforms, namely hCA VII and hCA I, which among the 15 human isoforms have been poorly investigated. hCA VII was the sole cytosolic isozyme, that at the beginning of this Ph.D thesis was not yet structurally characterized. This enzyme, similarly to hCA II, is a very efficient catalyst for hydration of carbon dioxide, being 10-50 times more active compared to other two cytosolic isoforms, hCA I and hCA XIII. However, in contrast to hCA II which is widely spread in human tissues, CA VII has a more limited distribution, being localized mainly in some brain tissues of humans and rats, in stomach, duodenum, colon, liver and skeletal muscle of mice. In the first part of my Ph.D thesis a complete biochemical characterization of this enzyme was carried out. Interestingly, these studies highlighted the capability of two reactive cysteines to be S-glutathionylated during the purification procedures. Since S-glutathionylation was reported in vivo also for another cytosolic CA isozyme, namely CA III, and was associated to a protective response to oxidative stress, this phenomenon was investigated in details also for CA VII. Such studies showed that Cys183 and Cys217 were involved in adduct formation. These reactive cysteines were mutagenized and the corresponding double mutant (C183S/C217S) was expressed. The native enzyme, its double mutant and the S-glutathionylated adduct were fully characterized for their CO2 hydration, esterase and phosphatase activity. These kinetic studies indicated that the modification of Cys183 and/or Cys217 by glutathione does not have a relevant impact on the active site of the enzyme, causing rather small differences in its specific activity. Moreover, an important observation was that hCA VII was highly effective as esterase and phosphatase, compared to other cytosolic CA isoforms, such as CA I, II, III and XIII. These findings seem to indicate that the observed S-glutathionylation, if present in vivo, is not involved in the regulation of the enzyme catalytic activity but rather, as observed for hCA III, can help hCA VII to function as an oxygen radical scavenger to protect cells from oxidative damage. Further in vivo studies are currently underway to investigate this issue. The X-ray crystallographic structure of a hCA VII mutated form in complex with a classical sulfonamide inhibitor, namely acetazolamide, was also solved. A detailed comparison of the obtained structure with those already reported for other CA isozymes provided novel insights into the catalytic properties of this protein family and offered the basis for a mutagenesis approach aimed at determining the contribution of the active site single residues to the enzyme catalytic efficiency. Moreover, on the basis of the structural differences detected within the active site of the various CA isoforms, further prospects for the design of isozyme-specific CA inhibitors have been obtained. hCA I was one of the first members of the CA family to be identified. Even though the 3D structure of this enzyme was first characterized already in 1975, only few structural studies on complexes formed with different inhibitors have been reported so far. Since these studies are fundamental for the drug design of isoform-selective inhibitors, part of this thesis has been dedicated to the structural characterization of a complex that hCA I forms with topiramate (TPM), which is a molecule of pharmacological interest for the treatment of epilepsy. The analysis of the structure of the complex showed that, upon binding of the inhibitor, the active site of hCA I undergoes a profound reorganization, which has never been observed in any other CA/inhibitor complex and might therefore be useful in designing CA inhibitors. Moreover, the comparison with hCA II/TPM and hCA VA/TPM complex structures, previously investigated, showed that a different H-bond network together with the movement of active site residues to accommodate the inhibitor may account for the difference of inhibition constants of TPM towards different CA isozymes. These data may be helpful in the design of CAIs selective for various isozymes.
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