Rodriguez-Lopez J N, Smith A T, Thorneley R N
Nitrogen Fixation Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom. Brighton, BN1 9QG, United Kingdom.
J Biol Chem. 1996 Feb 23;271(8):4023-30. doi: 10.1074/jbc.271.8.4023.
The observed pseudo-first order rate constant for the reaction between a horseradish peroxidase (HRP) variant (R38L)HRPC* and hydrogen peroxide saturates at high peroxide concentrations (Km = 11. 8 mm). The data are consistent with a two-step mechanism involving the formation of an HRP-H2O2 intermediate (k = 1.1 x 10(4) m-1 s-1) whose conversion to compound I is rate-limiting (k = 142 s-1) suggesting that Arg-38 is not only involved in the cleavage of the O-O bond of peroxide but also has an important role in facilitating the rapid binding of H2O2 to HRP. Rapid-scan spectrophotometry revealed the presence of a transient intermediate with a spectrum consistent with a ferric-hydroperoxy complex. At high peroxide concentrations (>500 microM), compound I is converted to compound III without the accumulation of compound II. Spectrophotometric titrations show that arginine 38 is also involved in modulating the apparent affinity of HRPC for reducing substrates such as guaiacol and p-cresol. The spectrum of the complex formed when these substrates bind to the ferric form of the mutant enzyme differs from that observed when they bind to the wild-type ferric enzyme. At neutral and alkaline pH compound I of (R38L)HRPC* was stable and reduced to ferric enzyme without apparent formation of compound II upon titration with p-cresol or ascorbic acid, suggesting a change in the rate-limiting step in the peroxidase cycle. Steady-state kinetic analyses carried out at pH 7.0 showed significant increases in the apparent Km for guaiacol, p-cresol, and 2, 2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS). The high stability of the oxyferryl form of (R38L)HRPC* and its low catalytic constant for reducing substrates also shows that arginine 38 modulates the reactivity of HRP compound I.
辣根过氧化物酶（HRP）变体（R38L）HRPC与过氧化氢反应的观测到的伪一级速率常数在高过氧化物浓度下达到饱和（Km = 11.8 mM）。数据与涉及形成HRP - H2O2中间体（k = 1.1×10⁴ m⁻¹ s⁻¹）的两步机制一致，该中间体向化合物I的转化是限速步骤（k = 142 s⁻¹），这表明精氨酸 - 38不仅参与过氧化物O - O键的断裂，而且在促进H2O2与HRP的快速结合中起重要作用。快速扫描分光光度法揭示了存在一种瞬态中间体，其光谱与铁 - 氢过氧络合物一致。在高过氧化物浓度（>500 μM）下，化合物I转化为化合物III，而没有化合物II的积累。分光光度滴定表明，精氨酸38也参与调节HRPC对还原底物如愈创木酚和对甲酚的表观亲和力。当这些底物与突变酶的铁形式结合时形成的复合物的光谱与它们与野生型铁酶结合时观察到的光谱不同。在中性和碱性pH下，（R38L）HRPC的化合物I是稳定的，在用对甲酚或抗坏血酸滴定时还原为铁酶，没有明显形成化合物II，这表明过氧化物酶循环中限速步骤发生了变化。在pH 7.0进行的稳态动力学分析表明，愈创木酚、对甲酚和2,2'-偶氮双（3-乙基苯并噻唑啉磺酸）（ABTS）的表观Km显著增加。（R38L）HRPC*的氧铁形式的高稳定性及其对还原底物的低催化常数也表明精氨酸38调节HRP化合物I的反应性。
