Single Turnover of Transient of Reactants Supports a Complex Interplay of Conformational States in the Mode of Action of Mycobacterium tuberculosis Enoyl Reductase

The enoyl reductase from Mycobacterium tuberculosis (MtInhA) was shown to be a major target for isoniazid, the most prescribed first-line anti-tuberculosis agent. MtInhA (EC 1.3.1.9) protein catalyzes hydride transfer 4S hydrogen of β-NADH carbon-3 long-chain 2-trans-enoyl thioester substrates (enoyl-ACP or enoyl-CoA) yield NAD+ and acyl-ACP acyl-CoA products. latter are long carbon chains meromycolate branch mycolic acids, which high-molecular-weight α-alkyl, β-hydroxy fatty acids mycobacterial cell wall. Here, stopped-flow measurements under single-turnover experimental conditions presented study transient reactants. Single-turnover experiments at various enzyme active sites were carried out. These studies suggested isomerization MtInhA:NADH binary complex in pre-incubation positive cooperativity that depends on number occupied by 2-trans-dodecenoyl-CoA (DD-CoA) substrate. Stopped-flow results burst analysis indicate product release does not contribute rate-limiting step MtInhA-catalyzed chemical reaction. bearings herein have function-based drug design discussed.