Kinetic and structural investigations of the allosteric site in human epithelial 15-lipoxygenase-2.
Kinetic and structural investigations of the allosteric site in human epithelial 15-lipoxygenase-2.
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人上皮 15-脂氧合酶-2 变构位点的动力学和结构研究。
DOI：
10.1021/bi9009242
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发表时间：
2009
期刊：
影响因子：
2.9
通讯作者：
Holman,TheodoreR
中科院分区：
文献类型：
--
作者：
Wecksler,AaronT;Kenyon,Victor;Garcia,NatalieK;Deschamps,JoshuaD;vanderDonk,WilfredA;Holman,TheodoreR
人类脂氧合酶（hLO）活性的变构调节最近被牵连在前列腺癌的细胞生物学。在目前的工作中，我们提出的同位素效应，pH值，和底物抑制剂的上皮细胞15-hLO-2的数据，探讨其机制行为的变构效应。15-hLO-2以AA和LA为底物时的Dkcat/Km较大，表明氢原子的夺取是主要的速率决定步骤，涉及两种底物的隧穿机制。对于AA，在高温和低温下都存在多个速率决定步骤（RDS），在高温下扩散和氢键重排都有贡献，但在低温下只有氢键重排有贡献。观察到的对氢键重排的动力学依赖性在加入变构效应物13-（S）-羟基十八碳二烯酸（13-HODE）后消除，并且在扩散或氢原子提取上没有观察到变构效应。观察到（kcat/KM）AA/（kcat/KM）LA比率具有pH依赖性，其与滴定曲线（pKa= 7.7）拟合，表明组氨酸残基的质子化，其可与13-HODE的羧酸盐形成氢键。假设这种相互作用，13-HODE对接到15-hLO-2同源性模型的溶剂暴露的组氨酸，发现与H627结合良好，表明变构位点的潜在位置。利用d31-LA作为抑制剂，证明d31-LA与变构位点的结合改变了15-hLO-2的构象，使得对底物的亲和力增加。该结果表明，变构结合将酶锁定到催化活性状态，这促进LA的结合并降低（kcat/KM）AA/（kcat/KM）LA比率。最后，15-hLO-2的13-HODEKD的大小比15-hLO-1的13-HODE的大小低200倍以上，使15-hLO-2的底物特异性变为1.9。这将改变LO产物分布并增加促肿瘤发生13-HODE的产生，可能代表13-HODE和15-hLO-2的促肿瘤发生反馈环。
Allosteric regulation of human lipoxygenase (hLO) activity has recently been implicated in the cellular biology of prostate cancer. In the current work, we present isotope effect, pH, and substrate inhibitor data of epithelial 15-hLO-2, which probe the allosteric effects on its mechanistic behavior. TheDkcat/KMfor 15-hLO-2, with AA and LA as substrate, is large indicating hydrogen atom abstraction is the principle rate-determining step, involving a tunneling mechanism for both substrates. For AA, there are multiple rate determining steps (RDS) at both high and low temperatures, with both diffusion and hydrogen bonding rearrangements contributing at high temperature, but only hydrogen bonding rearrangements contributing at low temperature. The observed kinetic dependency on the hydrogen bonding rearrangement is eliminated upon addition of the allosteric effector, 13-(S)-hydroxyoctadecadienoic acid (13-HODE), and no allosteric effects were seen on diffusion or hydrogen atom abstraction. The (kcat/KM)AA/(kcat/KM)LAratio was observed to have a pH dependence, which was fit with a titration curve (pKa= 7.7), suggesting the protonation of a histidine residue, which could hydrogen bond with the carboxylate of 13-HODE. Assuming this interaction, 13-HODE was docked to the solvent exposed histidines of a 15-hLO-2 homology model and found to bind well with H627, suggesting a potential location for the allosteric site. Utilizingd31-LA as an inhibitor, it was demonstrated that the binding ofd31-LA to the allosteric site changes the conformation of 15-hLO-2 such that the affinity for substrate increases. This result suggests that allosteric binding locks the enzyme into a catalytically competent state, which facilitates binding of LA and decreases the (kcat/KM)AA/(kcat/KM)LAratio. Finally, the magnitude of the 13-HODEKDfor 15-hLO-2 is over 200-fold lower than that of 13-HODE for 15-hLO-1, changing the substrate specificity of 15-hLO-2 to 1.9. This would alter the LO product distribution and increase the production of the pro-tumorigenic, 13-HODE, possibly representing a pro-tumorigenic feedback loop for 13-HODE and 15-hLO-2.