The Mycobacterium tuberculosis inhA-eneoded enoyl reductase has been proposed as the target of isoniazid, the most widely prescribed antitubercular drug. Isoniazid-dependent inactivation of the enoyl reductase has been suggested to require interaction of an activated form of the drug with enzymebound NADH. We have determined the binding of NADH to the wildtype enzyme using fluorescence titration, since the fluorescence of NADH is significantly enhanced upon binding to the enzyme. NADH binds tightly (Kd=0. 6μM) and stoichiometrically to the enzyme, and exhibits hyperbolic saturation. The S94A mutant has been previously identified from isoniazidresistant laboratory strains of M. smegmatis, and the I16T and V78A mutants have been identified from isoniazid-resistant clinical isolates of M. tuberculosis. These three mutants were prepared, expressed in E. coli and purified to homogeneity. All three mutants exhibited significantly lower affinities for NADH (Kd values ranged from 6-36#M), and displayed sigmoidal binding curves. These data support our proposal that inactivation of the enoyl reductase requires interaction of the activated drug with enzyme-bound NADH, and that isoniasid resistance can be accounted for by mutations which lead to decreases in the affinity for NADH.
|Original language||English (US)|
|Publication status||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology