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