Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction.
Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction.
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MutT 催化的三磷酸核苷焦磷酸水解酶反应的瞬态动力学研究。
DOI：
10.1021/bi0513599
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Mildvan,AlbertS
中科院分区：
文献类型：
--
作者：
Xia,Zuyong;Azurmendi,HugoF;Mildvan,AlbertS
MutT 焦磷酸水解酶在 Mg2+ 存在下，通过 Pβ 处的亲核取代催化三磷酸核苷水解，产生核苷酸和 PPi。 MutT 的最佳底物是诱变性 8-oxo-dGTP，其 Km 比 dGTP 低 540 倍。产物抑制研究提出了一种提议的单-双-异构动力学机制，其中 PPid 首先从酶-产物复合物 (k3) 解离，然后是 NMP (k4)，留下酶的产物结合形式 (F)，该形式在部分限速步骤 (k5) 中转化为底物结合形式 (E) [Saraswat, V., et al. (2002)生物化学 41, 15566−15577]。对 dGTP 和 8-oxo-dGTP 水解的单次和多次转换动力学研究以及数据与该机制的全局拟合已经产生了所有九个速率常数。与“iso”机制一致，dGTP 和 8-oxo-dGTP 水解的单周转研究显示底物结合的表观二级速率常数与它们的 kcat/Km 值相似，但远低于扩散极限 (∼109M-1s-1)：konapp= 7.2 × 104M-1s-1（对于 dGTP）和 konapp= 2.8 × 107M-1s-1（对于 dGTP） 8-氧代-dGTP。这些低konapp值是通过假设慢速iso步骤（k5 = 12.1 s-1）和底物结合的快速速率常数来拟合的：dGTP的k1 = 1.9 × 106M-1s-1和8-oxo-dGTP的k1 = 0.75 × 109M-1s-1（后者接近扩散极限）。以 dGTP 为底物，用 Mn2+ 取代 Mg2+ 不会改变 k1，这与第二球体 MutT−M2+−(H2O)−dGTP 复合物的形成一致，但会使 iso 步骤 (k5) 减慢 5.8 倍，而其反向 (k-5) 则减慢 25 倍，这表明 iso 步骤涉及金属配位的变化，可能是 Glu-53由酶结合金属使其可以起到通用碱的作用。 dGTP 和 8-oxo-dGTP 的多次周转研究显示产物形成的爆发，表明化学步骤 (k2) 之后的部分限速步骤。对于 dGTP，慢速步骤是化学步骤 (k2= 10.7 s-1) 和 iso 步骤 (k5= 12.1 s-1)。对于 8-oxo-dGTP，缓慢的步骤是 8-oxo-dGMP 产物的释放 (k4= 3.9 s-1) 和 iso 步骤 (k5= 12.1 s-1)，而化学步骤很快 (k2= 32.3 s-1)。瞬态动力学研究总体上与稳态kcat和Km值一致。速率常数和自由能图的比较表明，低浓度的 8-oxo-dGTP 是比 dGTP 更好的底物，因为它与 MutT 的结合速度快 395 倍，解离速度慢 46 倍，并且化学步骤快 3.0 倍。 MutT E 型底物的真实解离常数 (KD) 现在可以从 k-1/k1 获得，8-oxo-dGTP 为 3.5 nM，dGTP 为 62 μM，表明 8-oxo-dGTP 的结合比 dGTP 紧密 1.8 × 104 倍，对应于结合自由能低 5.8 kcal/mol。
The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pβ, to yield the nucleotide and PPi. The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of itsKmbeing 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PPidissociates first from the enzyme−product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V., et al. (2002)Biochemistry 41, 15566−15577]. Single- and multiple-turnover kinetic studies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism have yielded all of the nine rate constants. Consistent with an “iso” mechanism, single-turnover studies with dGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate binding similar to theirkcat/Kmvalues, but well below the diffusion limit (∼109M-1s-1):konapp= 7.2 × 104M-1s-1for dGTP andkonapp= 2.8 × 107M-1s-1for 8-oxo-dGTP. These lowkonappvalues are fitted by assuming a slow iso step (k5= 12.1 s-1) followed by fast rate constants for substrate binding:k1= 1.9 × 106M-1s-1for dGTP andk1= 0.75 × 109M-1s-1for 8-oxo-dGTP (the latter near the diffusion limit). With dGTP as the substrate, replacing Mg2+with Mn2+does not changek1, consistent with the formation of a second-sphere MutT−M2+−(H2O)−dGTP complex, but slows the iso step (k5) 5.8-fold, and its reverse (k-5) 25-fold, suggesting that the iso step involves a change in metal coordination, likely the dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base. Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partially rate-limiting steps following the chemical step (k2). With dGTP, the slow steps are the chemical step (k2= 10.7 s-1) and the iso step (k5= 12.1 s-1). With 8-oxo-dGTP, the slow steps are the release of the 8-oxo-dGMP product (k4= 3.9 s-1) and the iso step (k5= 12.1 s-1), while the chemical step is fast (k2= 32.3 s-1). The transient kinetic studies are generally consistent with the steady statekcatandKmvalues. Comparison of rate constants and free energy diagrams indicate that 8-oxo-dGTP, at low concentrations, is a better substrate than dGTP because it binds to MutT 395-fold faster, dissociates 46-fold slower, and has a 3.0-fold faster chemical step. The true dissociation constants (KD) of the substrates from the E-form of MutT, which can now be obtained fromk-1/k1, are 3.5 nM for 8-oxo-dGTP and 62 μM for dGTP, indicating that 8-oxo-dGTP binds 1.8 × 104-fold tighter than dGTP, corresponding to a 5.8 kcal/mol lower free energy of binding.