Evaluating the Impact of the Tyr158 pK_a on the Mechanism and Inhibition of InhA, the Enoyl-ACP Reductase from Mycobacterium tuberculosis

InhA, the Mycobacterium tuberculosis enoyl-ACP reductase, is a target for the tuberculosis (TB) drug isoniazid (INH). InhA inhibitors that do not require KatG activation avoid the most common mechanism of INH resistance, and there are continuing efforts to fully elucidate the enzyme mechanism to drive inhibitor discovery. InhA is a member of the short-chain dehydrogenase/reductase superfamily characterized by a conserved active site Tyr, Y158 in InhA. To explore the role of Y158 in the InhA mechanism, this residue has been replaced by fluoroTyr residues that increase the acidity of Y158 up to ∼3200-fold. Replacement of Y158 with 3-fluoroTyr (3-FY) and 3,5-difluoroTyr (3,5-F₂Y) has no effect on k_cat^app/K_M^app nor on the binding of inhibitors to the open form of the enzyme (K_i^app), whereas both k_cat^app/K_M^app and K_i^app are altered by seven-fold for the 2,3,5-trifluoroTyr variant (2,3,5-F₃Y158 InhA). ¹⁹F NMR spectroscopy suggests that 2,3,5-F₃Y158 is ionized at neutral pH indicating that neither the acidity nor ionization state of residue 158 has a major impact on catalysis or on the binding of substrate-like inhibitors. In contrast, K_i*^app is decreased 6- and 35-fold for the binding of the slow-onset inhibitor PT504 to 3,5-F₂Y158 and 2,3,5-F₃Y158 InhA, respectively, indicating that Y158 stabilizes the closed form of the enzyme adopted by EI*. The residence time of PT504 is reduced ∼four-fold for 2,3,5-F₃Y158 InhA compared to wild-type, and thus, the hydrogen bonding interaction of the inhibitor with Y158 is an important factor in the design of InhA inhibitors with increased residence times on the enzyme.