Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase
Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase
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DOI：
10.1021/acs.biochem.8b01167
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发表时间：
2019-02-12
期刊：
影响因子：
2.9
通讯作者：
Holman, Theodore
中科院分区：
文献类型：
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作者：
Aleem, Ansari Mukhtar;Tsai, Wan-Chen;Holman, Theodore
人血小板 ALOX12（hALOX12 或 h12-LOX）与多种人类疾病有关。本研究利用定点诱变研究了 hALOX12 的活性位点，以更彻底地了解它如何定位底物并实现近乎完美的区域特异性和立体特异性（即 12(S)-氢过氧化物产物的 100 +/- 5%）。具体来说，我们已经确定 Arg402 在底物结合中并不像之前在 hALOX15 中看到的那么重要，但 His596 可能在催化过程中锚定花生四烯酸的羧基末端方面发挥作用。此外，Phe414 与花生四烯酸双键 (Delta(11)) 产生 pi 堆积相互作用，Ala417/Val418 定义了空腔的底部。然而，Ala417/Val418 对 hALOX12 谱的影响明显小于 hALOX15 中的影响。将这两个残基突变为较大的氨基酸 (Ala417Ile/Val418Met) 仅将 15-HpETE 的生成增加了 24 +/- 2%，但相反，这些位置上较小的残基将 hALOX15 转化为几乎 100% hALOX12 反应性 [Gan et al. 2017]。 (1996) J.Biol。化学。 271、25412-254181。然而，通过使用 Ala417Ile/Val418Met/Ser594Thr 突变限制活性位点的宽度，我们能够将 15-HpETE 增加到 46 +/- 3%，这表明活性位点的深度和宽度都很重要。最后，残基 Leu407 在正确定位底物方面发挥着关键作用，如单个 Leu407Gly 突变体的 15-HpETE 增加至 21 +/- 1% 所示。这些结果概述了 hALOX12 相对于 hALOX15 的活性位点要求之间的关键差异，并解释了它们的产品特异性和抑制差异。
Human platelet ALOX12 (hALOX12 or h12-LOX) has been implicated in a variety of human diseases. The present study investigates the active site of hALOX12 to more thoroughly understand how it positions the substrate and achieves nearly perfect regio- and stereospecificities (i.e., 100 +/- 5% of the 12(S)-hydroperoxide product), utilizing site-directed mutagenesis. Specifically, we have determined that Arg402 is not as important in substrate binding as previously seen for hALOX15 but that His596 may play a role in anchoring the carboxy terminal of the arachidonic acid during catalysis. In addition, Phe414 creates a pi-stacking interaction with a double bond of arachidonic acid (Delta(11)), and Ala417/Val418 define the bottom of the cavity. However, the influence of Ala417/Val418 on the profile is markedly less for hALOX12 than that seen in hALOX15. Mutating these two residues to larger amino acids (Ala417Ile/Val418Met) only increased the generation of 15-HpETE by 24 +/- 2%, but conversely, smaller residues at these positions converted hALOX15 to almost 100% hALOX12 reactivity [Gan et al. (1996) J. Biol. Chem. 271, 25412-254181. However, we were able to increase 15-HpETE to 46 +/- 3% by restricting the width of the active site with the Ala417Ile/Val418Met/Ser594Thr mutation, indicating both depth and width of the active site are important. Finally, residue Leu407 is shown to play a critical role in positioning the substrate correctly, as seen by the increase of 15-HpETE to 21 +/- 1% for the single Leu407Gly mutant. These results outline critical differences between the active site requirements of hALOX12 relative to hALOX15 and explain both their product specificity and inhibitory differences.