THE ENZYME SHIKIMATE KINASE FROM MYCOBATERIUM TUBERCULOSIS AS A TARGET FOR DRUG DEVELOPMENT

Leonardo Astolfi Rosado (CPBMF); Luiz Augusto Basso (CPBMF); Diógenes Santiago Santos (CPBMF)

Tuberculosis is a worrisome disease due to its high infectious and mortality rates, reporting 9.2 million new cases and 1.7 million deaths each year. The shikimate pathway is essential for many organisms including M. tuberculosis (MTB), being responsible for the synthesis of aromatic amino acids, folates and ubiquinone. Shikimate kinase (E.C 2.7.1.71) catalyses the fifth reaction of the pathway, converting adenosine triphosphate (ATP) and shikimate to shikimate-3-phosphate and adenosine diphosphate. MTB constitutes a great problem to human health and the results of this work can be a great step to the development of agents against this pathogen. In the present work, experiments of binding were assayed with the intention of elucidating M. tuberculosis shikimate kinase (MtSK) enzymatic mechanism. Fluorescence titration was carried out with shikimate and shikimate-3-phosphate, substrate and product, respectively, and equilibrium dialysis titration was carried out to determine the interaction of ATP and ADP, substrate and product, respectively. The quenching in protein fluorescence upon shikimate and shikimate-3-phosphate binding to MtSK monomer was monitored to determine the dissociation constant at equilibrium for MtSK-shikimate and MtSK-shikimate-3-phosphate binary complex formation. A plot of shikimate concentration versus protein fluorescence variation upon binary complex formation shows an hyperbolic increase, yielding values of K_D = 124 ± 7 µM; a plot of shikimate-3-phosphate concentration versus protein fluorescence variation upon binary complex formation also shows an hyperbolic increase, yielding values of K_D = 162 ± 37 µM. Due the great inner filter effect observed on the spectrofluorimetric assay,  equilibrium dialysis and a spectrofluorimetric assay with an ATP analogue (ATP-Alexa Fluor 647) were carried out to determine the binding between the shikimate kinase monomer and ATP or ADP. The assays did not show a specific binding between the protein and the ligands. The data acquired indicates a Steady-State Ordered Bi Bi System mechanism, since in the absence of shikimate, ATP cannot bind in the activity site, and in the absence of shikimate-3-phosphate, ADP cannot bind to the enzyme. Soaking and (co-)crystallization experiments indicate a random sequence of substrate binding, a synergism between the substrates and a sequential mechanism to the release of the products, liberating ADP first and then shikimate-3-phosphate, suggesting two different routes of substrate binding; one of the proposed routes corroborates the experimental data produced here, proposing a binding of shikimate, generating a conformational change enhancing the binding of ATP, which agrees with previous works proposing a conformational change on the catalytic site without the presence of the substrate impairing the binding of ATP and its hydrolysis.