Jones G D, Russell L, Darley-Usmar V M, Stone D, Wilson M T
Department of Biological and Chemical Sciences, University of Essex, Colchester, United Kingdom.
Biochemistry. 1996 Jun 4;35(22):7197-203. doi: 10.1021/bi952425h.
We have used stopped-flow rapid reaction methods, employing both fluorescence and absorbance monitoring, together with HPLC analysis of the products to study the activation of soybean 15-lipoxygenase by 13(S)-hydroperoxy-9, 11(E,Z)-octadecadienoic acid (13-HPOD). When lipoxygenase is mixed with an equimolar concentration of 13-HPOD, the enzyme undergoes a rapid change in fluorescence. The rate of the change of fluorescence is dependent on the concentration of the 13-HPOD (k = 6.7 x 10(6) M-1 s-1) and is accompanied by activation of the enzyme. The fluorescence change is not accompanied by any change in the UV absorbance of the 13-HPOD, suggesting no loss of the conjugated diene during enzyme activation, and HPLC analysis of the products of the reaction confirms that the 13-HPOD can be recovered unchanged following this reaction. In the presence of an inhibitor (BWA4C, a hydroxamate inhibitor) that reduces the active-site iron, the 13-HPOD and the inhibitor are destroyed in a peroxidase-like reaction. On the basis of these observations we propose that 13-HPOD binds to the enzyme and facilitates activation of the enzyme, possibly through the formation of a protein radical, and that the 13-HPOD is not changed chemically in this process.
我们运用了停流快速反应方法，结合荧光和吸光度监测，并对产物进行高效液相色谱（HPLC）分析，以研究13(S)-氢过氧-9,11(E,Z)-十八碳二烯酸（13-HPOD）对大豆15-脂氧合酶的激活作用。当脂氧合酶与等摩尔浓度的13-HPOD混合时，酶的荧光会迅速发生变化。荧光变化的速率取决于13-HPOD的浓度（k = 6.7 x 10(6) M-1 s-1），并且伴随着酶的激活。荧光变化并未伴随13-HPOD的紫外吸光度发生任何变化，这表明在酶激活过程中共轭二烯没有损失，对反应产物的HPLC分析证实，在此反应后13-HPOD可以原样回收。在存在降低活性位点铁的抑制剂（BWA4C，一种异羟肟酸酯抑制剂）的情况下，13-HPOD和抑制剂在类似过氧化物酶的反应中被破坏。基于这些观察结果，我们提出13-HPOD与酶结合并促进酶的激活，可能是通过形成蛋白质自由基，并且在此过程中13-HPOD的化学性质没有改变。
