Ghiladi Reza A, Cabelli Diane E, Ortiz de Montellano Paul R
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280, USA.
J Am Chem Soc. 2004 Apr 21;126(15):4772-3. doi: 10.1021/ja031728t.
To gain insight into the mechanism of INH activation by KatG and to understand how resistance is conferred by the single active-site point mutation of KatG(S315T), we have employed pulse radiolysis as the means to initiate a catalytic pathway capable of mimicking the in vivo oxidation of isoniazid (INH). Radiolysis of a solution containing WT KatG revealed two intermediates: compound III (oxyferrous KatG) [415 (Soret), 545, 580 nm] formed [k1 = (4.47 +/- 0.91) x 105 M-1 s-1] in the absence of INH and compound II (410 (Soret), 540, 575 nm) formed [k1 = (4.43 +/- 0.69) x 105 M-1 s-1] in the presence of INH, with a comparison of the rates suggesting that compound III (rate-limiting) precedes compound II formation. By contrast, radiolysis of KatG(S315T) only led to compound III formation, whether INH was present [k1 = (4.72 +/- 0.99) x 105 M-1 s-1] or not [k1 = (4.51 +/- 1.38) x 105 M-1 s-1]. HPLC studies to determine the rates of INH-NADH adduct formation (an inhibitor of InhA) as catalyzed by KatG were also performed employing various oxidants: air [WT: (7.18 +/- 1.25) x 10-4, S315T: (0.74 +/- 0.39) x 10-4], superoxide (SOTS-1) [WT: (9.22 +/- 1.10) x 10-4, S315T: not detected], and tert-butylhydroperoxide [WT: (20.5 +/- 1.13) x 10-4, S315T: (10.15 +/- 0.19) x 10-4]. Taken together, the results from the pulse radiolysis work as well as the InhA inhibitor studies allow us to propose a mechanism capable of correlating the inability for the oxyferrous intermediate of KatG(S315T) to oxidize ("activate") INH to the suppressed formation of the INH-NADH adduct, thereby leading to INH resistance in Mycobacterium tuberculosis.
为深入了解KatG激活异烟肼（INH）的机制，并理解KatG（S315T）的单个活性位点点突变如何赋予耐药性，我们采用脉冲辐解作为引发催化途径的手段，该途径能够模拟体内异烟肼（INH）的氧化过程。对含有野生型KatG的溶液进行辐解，发现了两种中间体：在不存在INH的情况下形成化合物III（氧亚铁KatG）[415（Soret）、545、580 nm] [k1 = (4.47 ± 0.91) x 105 M-1 s-1]，在存在INH的情况下形成化合物II（410（Soret）、540、575 nm）[k1 = (4.43 ± 0.69) x 105 M-1 s-1]，速率比较表明化合物III（限速步骤）先于化合物II的形成。相比之下，无论是否存在INH，KatG（S315T）的辐解仅导致化合物III的形成[k1 = (4.72 ± 0.99) x 105 M-1 s-1] [k1 = (4.51 ± 1.38) x 105 M-1 s-1]。还使用各种氧化剂进行了HPLC研究，以确定KatG催化的INH-NADH加合物形成速率（InhA的抑制剂）：空气[野生型：(7.18 ± 1.25) x 10-4，S315T：(0.74 ± 0.39) x 10-4]、超氧化物（SOTS-1）[野生型：(9.22 ± 1.10) x 10-4，S315T：未检测到]和叔丁基过氧化氢[野生型：(20.5 ± 1.13) x 10-4，S315T：(10.15 ± 0.19) x 10-4]。综合来看，脉冲辐解研究以及InhA抑制剂研究的结果使我们能够提出一种机制，该机制能够将KatG（S315T）的氧亚铁中间体无法氧化（“激活”）INH与INH-NADH加合物形成受抑制相关联，从而导致结核分枝杆菌对INH产生耐药性。
