Uridina monofosfato quinase (EC 2.7.4.22) de Mycobacterium tuberculosis como alvo para desenvolvimento de drogas

• Ano de defesa: 2010
• Autor(a) principal: Rostirolla, Diana Carolina
• Orientador(a): Santos, Diógenes Santiago
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Pontifícia Universidade Católica do Rio Grande do Sul; Porto Alegre
• Palavras-chave em Português: BIOLOGIA MOLECULAR; BIOLOGIA CELULAR; NUCLEOTÍDEOS; DROGAS - PESQUISAS; TUBERCULOSE
• Link de acesso: http://hdl.handle.net/10923/1385

Tuberculosis (TB) remains a leading infectious killer worldwide and its causative agent, Mycobacterium tuberculosis, infects one third of the world population. The HIV co-infection and the emergence of multidrug and extensivelyresistant TB have provided a very alarming challenge to global health and led us to focus on the research for new and more effective therapeutics against the disease. Pyrimidine nucleotides are essential for many biochemical reactions and its synthesis constitutes an important step in the progression of TB. Therefore, the protein UMP kinase from M. tuberculosis (MtUMPK), which catalyses the phosphorylation of UMP to UDP and does not resemble its eukaryotic counterparts, is a promising target for the rational antitubercular drug design. In the present work, we report cloning of the pyrH gene coding region, heterologous recombinant protein in E. coli and purification to homogeneity. Moreover, we confirmed MtUMPK identity and its biological activity. Size exclusion chromatography showed that the protein is a tetramer in solution and kinetic studies revealed an allosteric behavior, suggesting that MtUMPK participates in the regulation of purine versus pyrimidine biosynthesis. Isothermal titration calorimetry (ITC) experiments showed that catalysis proceeds by a sequential ordered mechanism, in which the UMP substrate binds to the enzyme after the addition of ATP molecule, followed by a random displacement of ADP and UDP products.