A enzima chiquimato quinase (EC 2.7.1.71) de Mycobacterium tuberculosis como alvo para o desenvolvimento de drogas

• Ano de defesa: 2011
• Autor(a) principal: Rosado, Leonardo Astolfi
• 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: MEDICINA; FARMACOLOGIA; BIOQUÍMICA; BIOLOGIA MOLECULAR; ENZIMAS; TUBERCULOSE
• Link de acesso: http://hdl.handle.net/10923/4360

Tuberculosis (TB) still remains as one of main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), the fifth enzyme of the shikimate pathway, catalyzes phosphate transfer from ATP to the carbon-3 hydroxyl group of shikimate, yielding shikimate 3-phosphate and ADP. Disruption of aroK gene has demonstrated that MtSK is essential for the viability of M. tuberculosis. Here we present purification of MtSK to homogeneity, mass spectrometry analysis, N-terminal amino acid sequencing, and oligomeric state determination of the recombinant protein. Steadystate kinetics, and studies on ligand binding by fluorescence spectroscopy and isothermal titration calorimetry (ITC) suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release in which shikimate-3-phosphate release is followed by ADP dissociation to yield free enzyme. ITC results showed significant heat changes upon ligand binding to free MtSK enzyme, thereby providing thermodynamic signatures of non-covalent interactions to each binding process. These results provide a better understanding of the mode of action of MtSK that should be useful to guide the rational design of inhibitors of this enzyme that can be further be evaluated as antiTB drugs.