Avaliação funcional da enzima conversora de Angiotensina I (ECA) através de mutações sítio- dirigidas

• Ano de defesa: 2016
• Autor(a) principal: Pereira, Larissa Miranda [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Angiotensin converting enzyme; Structure; Enzima conversora de angiotensina; Mutação sítio-dirigida; Estrutura
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3669832; https://repositorio.unifesp.br/handle/11600/46224

Hypertension is a worldwide health problem that is emerging in the population as a major public health challenges and considered a major risk factor for cardiovascular and renal disease. Several studies have focused on the renin-angiotensin system, which is described as an endocrine system, where renin, an aspartyl endopeptidase, cleaves angiotensinogen in the Leu10-Val11 bond and releases the decapeptide angiotensin I (AI), which is converted to angiotensin II (AII), by the action of angiotensin-converting enzyme (ACE). ACE is an enzyme target of several therapeutic studies, and thus, the search for better understanding of its structure is the goal of many researchers. Thus, this study aims to investigate the general role of amino acid residues in the ACE inhibitors captopril and lisinopril interaction with the site located in the N-terminal protein shown during the studies of the structure of the N-domain isoforms of ACE, as well as the role of these amino acids on the stability and functionality of the enzyme. Thus, in this project the ACE?s amino acids residues targets were Ala361, Thr378 and Phe467, strong candidates for the interaction with the ACE inhibitors. In this work, we altered the amino acid residues to alanine and analyzed the effect of this mutation in relation to the processes of ligand-enzyme interaction and functional activity. The Ala361was mutated to tryptophan to verify the spatial importance of this residue in the enzyme. As a result, we obtained three enzymes with the region correct changed that were separately purified and one pool was obtained from the purification process for each enzyme, all of them having ACE specific activity, with a very similar profile of structure that is proposed in the literature. Even though similar to wild type ACE, the mutant enzymes showed differences in the interaction with specific substrates and inhibitors, demonstrating that these proposals regions are important for the interaction with substrate and inhibitors, emphasizing the importance of the N-domain isoform, considered a possible genetic hypertension marker.