Role of induced Fit in Limiting Discrimination against AZT by HIV Reverse Transcriptase
Role of induced Fit in Limiting Discrimination against AZT by HIV Reverse Transcriptase
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DOI：
10.1021/bi200204m
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发表时间：
2011-06-07
期刊：
影响因子：
2.9
通讯作者：
Johnson, Kenneth A.
中科院分区：
文献类型：
--
作者：
Kellinger, Matthew W.;Johnson, Kenneth A.
使用荧光标记的HIV逆转录酶（RT）进行单周转动力学研究，以评估核苷酸诱导的酶结构变化对AZT的选择性的作用，以探讨为什么AZT耐药形式的酶不能显着歧视AZT。荧光标记的HIV RT提供了一个信号，以监测异构化从“开放”到“关闭”的状态后，核苷酸结合。我们测量0核苷酸结合和酶异构化TTP和AZT-三磷酸的速率常数由野生型和AZT-耐药形式的酶含有胸苷类似物突变（TAMs）。我们表明，相对于野生型酶，TAM通过削弱基态核苷酸结合并降低化学反应速率来改变AZT掺入的动力学。然而，由TAMs HIV RT掺入AZT的较慢速率被较低的K（mj）抵消，这是由构象变化步骤的平衡引起的。相反，野生型酶的K（m）反映了结合速率和掺入速率之间的平衡，因此构象变化步骤不会达到平衡。这些数据再次表明，底物释放的速率，限制了逆转的底物诱导的构象变化，是关键的决定因素的作用，诱导适合酶的特异性。导致掺入速率较慢的突变具有通过允许构象变化步骤达到平衡而降低K（m）值的不幸后果。
Single turnover kinetic studies were conducted using fluorescently labeled HIV reverse transcriptase (RT) to evaluate the role of nucleotide-induced changes in enzyme structure in the selectivity against AZT in order to explore why AZT-resistant forms of the enzyme fail to significantly discriminate against AZT. Fluorescent labeling of HIV RT provided a signal to monitor the isomerization from "open" to "closed" states following nucleotide binding. We measured 0 the rate constants governing nucleotide binding and enzyme isomerization for TTP and AZT-triphosphate by the wild-type and AZT-resistant forms of the enzyme containing the thymidine analogue mutations (TAMs). We show that the TAMs alter the kinetics of AZT incorporation by weakening ground-state nucleotide binding and decreasing the rate of chemistry relative to the wild-type enzyme. However, the slower rate of incorporation of AZT by the TAMs HIV RT is counterbalanced a lower K(mj), resulting from the equilibration of the conformational change step. In contrast, the K(m) for the wild-type enzyme reflects the balance between rates of binding and incorporation so the conformational change step does not come to equilibrium. These data once again demonstrate that the rate of substrate release, limited by the reverse of the substrate-induced conformational change, is the key determinant of the role of induced fit in enzyme specificity. Mutations leading to slower rates of incorporation have the unfortunate consequence of lowering the K(m) value by allowing the conformational change step to come to equilibrium.