Butovich I A, Reddy C C
Center for Molecular Toxicology, Environmental Resources Research Institute, Pennsylvania State University, 115 Henning Building, University Park, PA 16802, USA.
Biochim Biophys Acta. 2001 Apr 7;1546(2):379-98. doi: 10.1016/s0167-4838(01)00162-5.
It was shown for the first time that potato tuber lipoxygenase (ptLOX) catalyzed the aerobic oxidation of 1-monolinoleoyl-rac-glycerol (mLG) in a mixed micellar reaction solution with the non-ionic detergent monododecyl ether of decaoxyethylene glycol. No hydrolysis of mLG occurred during the reaction. The four major reaction products obtained at 23 degrees C were identified as 1-[9-hydroperoxy-10E,12Z-octadecadienoyl]-rac-glycerol (9-(E,Z)HPODE-GE, 41%), 1-[13-hydroperoxy-9Z,11E-octadecadienoyl]-rac-glycerol (13-(Z,E)-HPODE-GE, 17%), and their all-trans isomers ( approximately 21% each). The molar fraction of all-trans isomers depended on the temperature of the reaction solution; it was found that at 0 degrees C their molar fractions were approximately 15.5% each, while 9-(E,Z)HPODE-GE and 13-(Z,E)-HPODE-GE gave 42% and 27%, respectively, of the overall product. A free radical scavenger, 4-hydroxy-TEMPO, dramatically increased the molar fraction of 9-(E,Z)HPODE-GE, yielding 83% at 23 degrees C, at the expense of all other products. Chiral HPLC of 9-(E,Z)HPODE-GE formed in the presence of 4-hydroxy-TEMPO revealed that it was composed of approximately 94% S and approximately 6% (R) isomers. This assures largely a uniform orientation of mLG molecules in the ptLOX active center, with their methyl end most likely deepened into the protein globule. The second major product, 13-(Z,E)-HPODE-GE, which yielded approximately 9% of the total product formed in the presence of 4-hydroxy-TEMPO, was racemic, and so were the all-trans isomers. Therefore, the last three cannot be considered the true products of the enzyme reaction, which is known to be stereospecific. It appears that they were formed as a result of (i) leakage of the pentadienyl radicals from the ptLOX active center and their subsequent non-enzymatic dioxygenation, and/or (ii) leakage of the peroxyl radicals leading to a free radical chain reaction affording all positional, geometrical and stereoisomers of the products. This reaction resembles ptLOX oxidation of another non-ionizable substrate, linoleyl alcohol [I.A. Butovich, S.M. Luk'yanova, C.C. Reddy, Arch. Biochem. Biophys. 378 (2000) 65-77], and differed substantially from oxidation of ionizable linoleic acid. Consequently, formation of large amounts of the non-specific oxidation products might be considered a universal characteristic of ptLOX oxidation of non-ionizable compounds.
首次发现，在含有十聚乙二醇单十二烷基醚这种非离子型去污剂的混合胶束反应溶液中，马铃薯块茎脂氧合酶（ptLOX）催化1-单亚油酰-消旋甘油（mLG）的需氧氧化反应。反应过程中未发生mLG的水解。在23℃下获得的四种主要反应产物被鉴定为1-[9-氢过氧-10E,12Z-十八碳二烯酰]-消旋甘油（9-(E,Z)HPODE-GE，41%）、1-[13-氢过氧-9Z,11E-十八碳二烯酰]-消旋甘油（13-(Z,E)-HPODE-GE，17%）及其全反式异构体（各约21%）。全反式异构体的摩尔分数取决于反应溶液的温度；发现在0℃时，它们的摩尔分数各约为15.5%，而9-(E,Z)HPODE-GE和13-(Z,E)-HPODE-GE分别占总产物的42%和27%。自由基清除剂4-羟基-TEMPO显著提高了9-(E,Z)HPODE-GE的摩尔分数，在23℃时达到83%，代价是其他所有产物减少。在4-羟基-TEMPO存在下形成的9-(E,Z)HPODE-GE的手性高效液相色谱分析表明，它由约94%的S异构体和约6%的(R)异构体组成。这在很大程度上确保了mLG分子在ptLOX活性中心的取向一致，其甲基端很可能深入到蛋白质球状体中。第二种主要产物13-(Z,E)-HPODE-GE在4-羟基-TEMPO存在下形成的总产物中约占9%，它是外消旋的，全反式异构体也是如此。因此，后三种产物不能被视为该酶反应的真正产物，因为已知该酶反应具有立体特异性。似乎它们是由于（i）戊二烯基自由基从ptLOX活性中心泄漏并随后进行非酶双加氧反应，和/或（ii）过氧自由基泄漏导致自由基链反应，从而产生产物的所有位置、几何和立体异构体而形成的。该反应类似于ptLOX对另一种不可电离底物亚油醇的氧化反应[I.A. Butovich, S.M. Luk'yanova, C.C. Reddy, Arch. Biochem. Biophys. 378 (2000) 65-77]，与可电离的亚油酸的氧化反应有很大不同。因此，大量非特异性氧化产物的形成可能被认为是ptLOX对不可电离化合物氧化的一个普遍特征。
