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. 2003 Nov 25;100(24):13881-6.
doi: 10.1073/pnas.2235848100. Epub 2003 Nov 17.

The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance

Richa Rawat  1 , Adrian Whitty, Peter J Tonge
Affiliations

The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance

Richa Rawat et al. Proc Natl Acad Sci U S A. .

Abstract

Isoniazid (INH), a frontline antitubercular drug, inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis, by forming a covalent adduct with the NAD cofactor. Here, we report that the INH-NAD adduct is a slow, tight-binding competitive inhibitor of InhA. Demonstration that the adduct binds to WT InhA by 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 Ki = 0.75 +/- 0.08 nM, reconciles 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* (k2 = 0.13 +/- 0.01 min(-1)) is similar to the maximum rate constant observed for InhA inhibition in reaction mixtures containing InhA, INH, NADH, and the INH-activating enzyme KatG (catalase/peroxidase from M. tuberculosis), 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, I21V, I47T, 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. Finally, for M161V, an InhA mutation that correlates with resistance to the common biocide triclosan in Mycobacterium smegmatis, binding to form the initial EI complex is significantly weakened, explaining why this mutant inactivates more slowly than WT InhA when incubated with INH, NADH, and KatG.

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Figures

Scheme 1.
Scheme 1.
Scheme 2.
Scheme 2.
Scheme 3.
Scheme 3.
Fig. 1.
Fig. 1.
(A) Time-dependent inactivation of WT InhA by 0–80 nM INH-NAD. The solid curves represent the best fit of the data to Eq. 1 for slow binding inhibition. (B) kobs from A plotted as a function of [I]. Data are fitted by using Eq. 2 to give values for k2, k–2, and formula image. (C) Initial velocity (νi) from A plotted as a function of [I]. Data are fitted by using Eq. 3 to give a value for formula image. (D) Ratio of νs and νi from A plotted as a function of [I]. Data are fitted by using Eq. 4 to give a value for formula image.
Fig. 2.
Fig. 2.
Free-energy profile for the interaction of INH-NAD with InhA. formula image was calculated for the EI and EI* states by using K–1 and Ki and applying a standard state of 1 μM. The free energy of the transition state for the conversion of EI to EI* (‡2) is determined from k2.(A) Free-energy profile for WT InhA. (B) Free-energy profiles for WT enzyme (heavy line) and M161V (light line, ⋄), and data points for I21V (▿), I47T (○), and S94A (□).
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