Mechanism of reaction of hydrogen peroxide with horseradish peroxidase:: Identification of intermediates in the catalytic cycle
Mechanism of reaction of hydrogen peroxide with horseradish peroxidase:: Identification of intermediates in the catalytic cycle
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DOI：
10.1021/ja011853
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发表时间：
2001-12-05
影响因子：
15
通讯作者：
Thorneley, RNF
中科院分区：
文献类型：
--
作者：
Rodríguez-López, JN;Lowe, DJ;Thorneley, RNF
利用电子吸收、快速扫描停流和电子顺磁共振（EPR）等方法，研究了辣根过氧化物酶同工酶C （HRPC）与过氧化氢在酸性和碱性条件下形成活性酶中间化合物I的反应机理。利用定点突变酶His42—> Leu （H42L）研究了活性位点残基His42和Arg38在控制H2O2氧-氧键异裂中的作用。Arg38—> Leu （R38L）和Arg38—>gly （R38G）。H42L与H2O2的双相反应动力学表明存在中间产物，在酸性pH下，可能由于中性酶H2O2配合物和含铁-过氧阴离子的化合物0而发生可逆的第二步反应。EPR还表明，在距离血红素铁10埃以上的地方形成了一个蛋白质自由基。H42L/ H2O2反应产物与2,2′-氮唑（3-乙基苯并噻唑啉磺酸）（ABTS）反应的化学计量学与浓度有关，在0.7 mM ABTS以上，其值从2降至1。这些数据可以解释H2O2在H42L中发生均溶裂解。与野生型HRPC相比，H42L形成化合物I的表观速率虽然低，但与pH无关，野生型HRPC的速率在酸性pH下下降，这表明可电离基团的参与与pK(a)接近4。在R38L和R38G中，表观pK(a)向类似于8的方向移动，但没有证据表明H2O2发生同溶裂解。这些数据表明，在中性pH下，His42首先作为质子受体（碱催化剂），然后作为供体（酸催化剂），并预测了在酸性pH下观察到的异裂速率较慢和效率较低。Arg38对降低His42的pKa有影响，并且对活性位点的H2O2有定向作用，但对质子转移没有直接作用。
The mechanism of the reaction of horseradish peroxidase isoenzyme C (HRPC) with hydrogen peroxide to form the reactive enzyme intermediate compound I has been studied using electronic absorbance, rapid-scan stopped-flow, and electron paramagnetic resonance (EPR) spectroscopies at both acid and basic pH. The roles of the active site residues His42 and Arg38 in controlling heterolytic cleavage of the H2O2 oxygen-oxygen bond have been probed with site-directed mutant enzymes His42 --> Leu (H42L), Arg38 --> Leu (R38L), and Arg38 --> Gly (R38G). The biphasic reaction kinetics of H42L with H2O2 suggested the presence of an intermediate species and, at acid pH, a reversible second step, probably due to a neutral enzyme H2O2 complex and the ferric -peroxoanion-containing compound 0. EPR also indicated the formation of a protein radical situated more than similar to 10 Angstrom from the heme iron. The stoichiometry of the reaction of the H42L/ H2O2 reaction product and 2,2 ' -azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) was concentration dependent and fell from a value of 2 to 1 above 0.7 mM ABTS. These data can be explained if H2O2 undergoes homolytic cleavage in H42L. The apparent rate of compound I formation by H42L, while low, was pH independent in contrast to wild-type HRPC where the rate falls at acid pH, indicating the involvement of an ionizable group with pK(a) approximate to 4. In R38L and R38G, the apparent pK(a) was shifted to similar to8 but there is no evidence that homolytic cleavage of H2O2 occurs. These data suggest that His42 acts initially as a proton acceptor (base catalyst) and then as a donor (acid catalyst) at neutral pH and predict the observed slower rate and lower efficiency of heterolytic cleavage observed at acid pH. Arg38 is influential in lowering the pKa of His42 and additionally in aligning H2O2 in the active site, but it does not play a direct role in proton transfer.