Caracterização bioquímica e estrutural da enzima inosina-uridina nucleosídeo hidrolase de leishmania braziliensis

• Ano de defesa: 2017
• Autor(a) principal: Dalberto, Pedro Ferrari
• 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
• Programa de Pós-Graduação: Programa de Pós-Graduação em Biologia Celular e Molecular
• Departamento: Escola de Ciências
• País: Brasil
• Palavras-chave em Português: Protozoário; Leishmania; Salvamento de Purinas; Hidrolase; IU-NH
• Área do conhecimento CNPq: CIENCIAS BIOLOGICAS::BIOLOGIA GERAL
• Link de acesso: http://tede2.pucrs.br/tede2/handle/tede/9057

Leishmaniasis is considered one of the main endemic diseases in the world, and Brazil is among the countries, which has the highes t incidence of cutaneous and mucocutaneous forms of leishmaniosis caused mainly by Leishmania braziliensis . Although the treatment of this disease is available, the first line drugs against leishmaniosis have several limitations: parenteral administration, long duration of treatment, and serious toxicities. One key metabolic characteristic of th is parasite is the lack of purine de novo biosynthesis pathway, relying only on the purine salvage pathway for nucleotide synthesis. The better understanding of the purine salvage pathway can reveal details of the biology of L. braziliensis that could be used to develop new strategies to combat this parasite. The inosine uridine nucleoside hydrolase from L. braziliensis Lb IU NH) plays an important role in the salvage process and is an attractive drug target as there is no similar catalytic activity in mammals. Here, it is described the cloning, expression, and purification of the homogenous recombinant protein. Determination of Lb IU NH steady state kinetic constants f or inosine, adenosine, cytidine and uridine are reported. These data suggest distinct functions and characteristics of a nonspecific hydrolase. Thermodynamic profile suggests that only ribose can bind to free enzyme with favorable enthalpic ( Δ H) and entropic ( Δ S) contributions T hermodynamic activation parameters ( E a , Δ G ##, Δ S ##, Δ H ##) for the Lb IU NH catalyzed chemical reaction, pre steady state kinetics, solvent kinetic isotope effects, and pH rate profiles are also presented. In addition, the crystal structure of Lb IU NH in complex with ribose and Ca 2+ at 1.5 Å resolution is described.