Enzyme-catalyzed and enzyme-triggered pathways in dioxygenation of 1-monolinoleoyl-rac-glycerol by potato tuber lipoxygenase

It was shown for the first time that potato tuber lipoxygenase (ptLOX) catalyzed the aerobic oxidation of l-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 degreesC 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,E3-HPODE-GE, 17%), and their all-trans isomers (similar to 21% each). The molar fraction of all-trans isomers depended on the temperature of the reaction solution; it was found that at 0 degreesC their molar fractions were similar to 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)HPODEGE, yielding 83% at 23 degreesC, 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 similar to 94% S and similar to6% (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 similar to9% 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. (C) 2001 Elsevier Science B.V. All rights reserved.