Mutants in the structural gene of the inhA-encoded NADH-dependent 2-trans enoyl-acyl carrier protein reductase were identified from isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. Recombinant InhA proteins with defined single amino acid replacements were expressed in Escherichia coli and purified to homogeneity. Steady-state kinetic parameters for wild type (WT) and I16T, I21V, I47T, and I95P mutants of the enoyl reductase were measured spectrophotometrically. NADH binding to WT and I16T, I21V, I47T, S94A, and I95P mutant reductases were determined by fluorescence spectroscopy and demonstrated that all mutant enzymes had reduced NADH affinity and that NADH binding to all mutants was cooperative as compared with the hyperbolic binding of NADH to the WT enzyme. Since KatG-produced electrophilic derivatives of isoniazid have been suggested to inactivate the enoyl reductase-NADH complex, the kinetics of inactivation for the WT and I21V and I95P mutants was determined. Both mutations resulted in significantly increased values for the apparent first-order rate constant of inactivation.