Basso L A, Zheng R, Musser J M, Jacobs W R, Blanchard J S
Department of Biochemistry and Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Infect Dis. 1998 Sep;178(3):769-75. doi: 10.1086/515362.
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.
从结核分枝杆菌的异烟肼耐药临床分离株中鉴定出inhA编码的NADH依赖性2-反式烯酰-酰基载体蛋白还原酶结构基因中的突变体。在大肠杆菌中表达具有确定的单氨基酸替换的重组InhA蛋白，并纯化至同质。通过分光光度法测量烯酰还原酶野生型（WT）以及I16T、I21V、I47T和I95P突变体的稳态动力学参数。通过荧光光谱法测定NADH与WT以及I16T、I21V、I47T、S94A和I95P突变体还原酶的结合，结果表明所有突变酶对NADH的亲和力均降低，并且与NADH与WT酶的双曲线结合相比，NADH与所有突变体的结合具有协同性。由于已表明KatG产生的异烟肼亲电衍生物可使烯酰还原酶-NADH复合物失活，因此测定了WT以及I21V和I95P突变体的失活动力学。这两种突变均导致失活的表观一级速率常数显著增加。
