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-ACPreductase, is a target for the tuberculosis (TB) drugisoniazid (INH). InhA inhibitors that do not require KatG activationavoid the most common mechanism of INH resistance, and there are continuingefforts to fully elucidate the enzyme mechanism to drive inhibitordiscovery. InhA is a member of the short-chain dehydrogenase/reductasesuperfamily characterized by a conserved active site Tyr, Y158 inInhA. To explore the role of Y158 in the InhA mechanism, this residuehas been replaced by fluoroTyr residues that increase the acidityof Y158 up to similar to 3200-fold.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 formof the enzyme (K (i) (app)), whereasboth 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-F(3)Y158 InhA). F-19 NMR spectroscopy suggeststhat 2,3,5-F(3)Y158 is ionized at neutral pH indicating thatneither the acidity nor ionization state of residue 158 has a majorimpact on catalysis or on the binding of substrate-like inhibitors.In contrast, K (i)*(app) is decreased6- and 35-fold for the binding of the slow-onset inhibitor PT504 to 3,5-F(2)Y158 and 2,3,5-F(3)Y158 InhA, respectively,indicating that Y158 stabilizes the closed form of the enzyme adoptedby EI*. The residence time of PT504 is reduced similar to four-foldfor 2,3,5-F(3)Y158 InhA compared to wild-type, and thus,the hydrogen bonding interaction of the inhibitor with Y158 is animportant factor in the design of InhA inhibitors with increased residencetimes on the enzyme.

Automated summary

By mutating the InhA enzyme, this study revealed the role of Y158 in the enzyme mechanism and found that it stabilizes the closed conformation of the enzyme, which is important for designing InhA inhibitors with increased residence times.