| Reemergence of tuberculosis (TB) has led to a renewed interest in elucidating the mode of action of well-known antitubercular drugs such as isoniazid (INH). INH, inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis , by forming a covalent adduct with the NAD cofactor. We have shown that INH-NAD adduct is a slow, tight binding competitive inhibitor of InhA. The adduct binds to wild-type (WT) InhA via a two-step enzyme inhibition mechanism, with initial, weak binding (K−1 = 16 ± 11 nM) followed by slow conversion to a final inhibited complex (EI*) with overall K i = 0.75 ± 0.08 nM, reconciling existing contradictory values for the inhibitory potency of INH-NAD for InhA. The first-order rate constant for conversion of the initial EI complex to EI* is similar to the maximum rate constant observed for InhA inhibition in reaction mixtures containing InhA, INH, NADH and the INH-activating enzyme KatG, consistent with an inhibition mechanism in which the adduct forms in solution rather than on the enzyme. Importantly, three mutations that correlate with INH resistance, 121V, 147T and S94A, have little impact on the inhibition constants. Thus, drug-resistance does not result simply from a reduction in affinity of INH-NAD for pure InhA. Instead, we hypothesize that protein-protein interactions within the FASII complex are critical to the mechanism of INH action. Moreover, for M161V, an InhA mutation that correlates with triclosan resistance in M. smegmatis, binding to form the initial EI complex is significantly weakened, explaining slower inactivation as compared to WT InhA when incubated with INH, NADH and KatG.; Additionally, to increase the understanding of the mechanism of InhA, the active site catalytic triad of InhA was probed. Residues F149 and Y158 of the catalytic triad exhibited important roles not only in catalysis but also in the interaction of triclosan and the INH-NAD adduct. These results will provide valuable information for designing improved enoyl reductase inhibitors based on the triclosan skeleton. |
