Department of Pathology and National Research Centre for Growth and Development, University of Otago Christchurch, P.O. Box 4345, Christchurch, New Zealand.
J Biol Chem. 2011 May 20;286(20):18048-55. doi: 10.1074/jbc.M111.232355. Epub 2011 Mar 8.
Peroxiredoxins (Prx) are thiol peroxidases that exhibit exceptionally high reactivity toward peroxides, but the chemical basis for this is not well understood. We present strong experimental evidence that two highly conserved arginine residues play a vital role in this activity of human Prx2 and Prx3. Point mutation of either ArgI or ArgII (in Prx3 Arg-123 and Arg-146, which are ∼3-4 Å or ∼6-7 Å away from the active site peroxidative cysteine (C(p)), respectively) in each case resulted in a 5 orders of magnitude loss in reactivity. A further 2 orders of magnitude decrease in the second-order rate constant was observed for the double arginine mutants of both isoforms, suggesting a cooperative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure suggest strong catalytic effects of H-bond-donating functional groups to the C(p) sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cation in the calculations to mimic the functional group of arginine, we were able to locate two transition structures that indicate rate enhancements consistent with our experimentally observed rate constants. Our results provide strong evidence for a vital role of ArgI in activating the peroxide that also involves H-bonding to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H(2)O(2) and may have wider implications for protein thiol reactivity toward peroxides.
过氧化物酶（Prx）是对过氧化物表现出极高反应性的硫醇过氧化物酶，但对此的化学基础还不是很了解。我们提出了强有力的实验证据，表明两个高度保守的精氨酸残基在人 Prx2 和 Prx3 的这种活性中起着至关重要的作用。在每种情况下，点突变 ArgI 或 ArgII（在 Prx3 的 Arg-123 和 Arg-146 中，分别距离活性位点过氧化物半胱氨酸（C（p））约 3-4 Å 或约 6-7 Å），反应性都降低了 5 个数量级。两种同工酶的双精氨酸突变体的二级速率常数进一步降低了 2 个数量级，表明这些残基具有协同功能。使用 G4 程序进行的详细从头计算表明，氢键供体官能团以协同方式对 C（p）硫和过氧化物的反应性和离去氧具有强烈的催化作用。在计算中使用胍阳离子来模拟精氨酸的官能团，我们能够定位两个过渡态结构，表明与我们观察到的实验速率常数一致的速率增强。我们的结果为 ArgI 在激活过氧化物中所起的重要作用提供了有力的证据，这也涉及到与 ArgII 的氢键。这种机制可以解释过氧化物酶对 H2O2 的异常反应性，并且可能对蛋白质巯基对过氧化物的反应性具有更广泛的影响。
