Efeitos do crowding macromolecular na atividade enzimática da 2-trans-ENOIL-ACP (COA) redutaze de Mycobacterium tuberculosis

Abstract

The cellular milieu is a complex and crowded aqueous solution. It is thus expected that this large concentration of macromolecules causes deviations from solution ideality. To mimic the intracellular environment, crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (Km, kcat, and kcat/Km) for the chemical reaction catalyzed by 2-trans-enoyl-ACP (CoA) from Mycobacterium tuberculosis (InhA). The results showed that ficoll 70, ficoll 400, and dextran 70 had negligible effects on InhA activity in the range of concentrations used. On the other hand, a complex effect was observed for PEG 6000. Sucrose, which was employed in control experiments, decreased both the kcat/Km values for NADH and kcat for 2-trans-dodecenoyl-CoA (DD-CoA) substrate in a concentration-dependent manner. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution, which likely accounts for the steady-state kinetic results. The presence of crowding agents appears to alter the relative abundance of different conformers of InhA in solution. The effects of the crowding agents on the energy (Ea and Eη), enthalpy (ΔH#), entropy (ΔS#), and Gibbs free energy (ΔG#) of activation were determined. The ΔG# values for all crowding agents tested were similar to dilute buffer, suggesting that excluded volume effects did not facilitate stable activated ES# complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that "soft" interactions may play a role in macromolecular crowding effects.