Kinetic Study of the Inhibition Mechanism of Dehaloperoxidase-Hemoglobin A by 4-Bromophenol
Kinetic Study of the Inhibition Mechanism of Dehaloperoxidase-Hemoglobin A by 4-Bromophenol
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DOI：
10.1021/jp3116353
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发表时间：
2013-07-18
影响因子：
3.3
通讯作者：
Franzen, Stefan
中科院分区：
文献类型：
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作者：
Zhao, Jing;Franzen, Stefan
用Micachis-Menten和暂态动力学分析方法研究了4-溴苯酚(4-BP)对脱氢过氧化物酶-血红蛋白(DHP)的抑制机理。使用停流技术在抑制剂浓度小于酶浓度10倍的情况下混合DHP和H_2O_2的暂态动力学表明，4-BP不能完全阻止H_2O_2进入远端口袋激活DHP。目前还不清楚在这些条件下是否形成了氧铁中间体，也不清楚DHP的4个碱基结合形式的H_2O_2反应可能有其他途径。通过奇异值分解(SVD)和全局拟合分析，在动态动力学实验中发现了两个新物种。不是形成非结合形式的化合物ES，而是观察到具有蓝移的Soret带和双峰Q带的抑制剂结合中间体。该中间体随后被转化为不同于在不受抑制的酶中形成的化合物RH的终点物种。为了从热力学角度理解酶的抑制机理，测定了DHP的缓蚀剂结合常数，进行了台式混合动力学研究。结果表明，在283K~298K温度范围内，点上的缓蚀常数K-(Nu)从2.56 mm下降到0.15 mm，由此可以确定缓蚀剂结合的热焓和熵分别为-135.5+/-20.9kJ/mAi和526.1+/-71.9J/(mol.K)，从而得出缓蚀剂结合是以熵驱动的结论。
The mechanism of dehaloperoxidase-hemoglobin (DHP) inhibition by 4-bromophenol (4-BP) was investigated using Michealis-Menten and transient-state kinetic analyses. Transient-state kinetics using the stopped-flow technique to mix DHP and H2O2 in the presence of inhibitor concentrations less than 10-fold greater than the enzyme concentration show that 4-BP does not fully impede H2O2 entering the distal pocket to activate DHP. It is not clear whether an oxoferryl intermediate is formed under these conditions and there may be alternative pathways for H2O2 reaction in the 4-BP bound form of DHP. Two new species have been identified during the reaction of 4-BP bound form of DHP in the transient-state kinetic experiment by using Singular Value Decomposition (SVD) and global-fitting analysis. Rather than forming Compound ES in the unbound form, an inhibitor bound intermediate that possesses blue-shifted Soret band and a double peaked Q-band is observed. This intermediate is subsequently converted to the end-point species that is distinguished from Compound RH formed in the uninhibited enzyme. Bench-top mixing kinetics of DHP were conducted in order to determine the inhibitor binding constant and to understand the enzyme inhibition mechanism from a thermodynamic perspective. It was found that the inhibition constant, K-(nu) over dot decreased from 2.56 mM to 0.15 mM over the temperature range from 283 to 298 K, which permits determination of the enthalpy and entropy for inhibitor binding as -135.5 +/- 20.9 kJ/mai and 526.1 +/- 71.9 J/(mol.K), respectively, leading to the conclusion that inhibitor binding is entropically driven.