在结核分枝杆菌烯酰还原酶的作用模式中,瞬态反应物的单一周转支持构象状态的复杂相互作用

Leonardo K. Martinelli, M. Rotta, C. V. Bizarro, Pablo Machado, L. A. Basso
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摘要

来自结核分枝杆菌的烯基还原酶(MtInhA)被证明是异烟肼的主要靶点,异烟肼是最常用的一线抗结核药物。MtInhA (EC 1.3.1.9)蛋白催化氢化物从β-NADH的4S氢转移到长链2-反式烯基硫酯底物(烯基- acp或烯基- coa)的碳-3,生成NAD+和酰基- acp或酰基- coa产物。后者是分枝杆菌细胞壁的高分子量α-烷基、β-羟基脂肪酸,是分枝杆菌细胞壁的长碳链菌酸分支。本文介绍了单次周转实验条件下的停流测量,用于研究反应物的瞬态。在不同的酶活性位点进行了单次翻转实验。这些研究表明,MtInhA:NADH二元复合物在孵育前的异构化和正协同性取决于2-反式十二烷基辅酶a (DD-CoA)底物占据的酶活性位点的数量。爆发分析的停流结果表明,产物释放对mtinha催化的化学反应的限速步骤没有贡献。本文的研究结果对基于功能的抗结核药物设计的影响进行了讨论。
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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. The MtInhA (EC 1.3.1.9) protein catalyzes the hydride transfer from the 4S hydrogen of β-NADH to carbon-3 of long-chain 2-trans-enoyl thioester substrates (enoyl-ACP or enoyl-CoA) to yield NAD+ 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 MtInhA:NADH binary complex in pre-incubation and positive cooperativity that depends on the number of enzyme active sites occupied by the 2-trans-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 MtInhA-catalyzed chemical reaction. The bearings that the results presented herein have on function-based anti-tuberculosis drug design are discussed.
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