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 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.
Butovich
I A
IA
Center for Molecular Toxicology, Environmental Resources Research Institute, Pennsylvania State University, 115 Henning Building, University Park, PA 16802, USA. iabl@psu.edu
Reddy
C C
CC
D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004951:Esterification; D016166:Free Radical Scavengers / Q000737:chemistry; D005989:Glycerides / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008084:Lipoxygenase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D011198:Solanum tuberosum / Q000201:enzymology; D012995:Solubility / Q000187:drug effects; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
C426011:1-(13-hydroperoxy-9,11-octadecadienoyl)glycerol; C426010:1-(9-hydroperoxy-10,12-octadecadienoyl)glycerol; D003902:Detergents; D016166:Free Radical Scavengers; D005989:Glycerides; D012967:Sodium Dodecyl Sulfate; D008084:Lipoxygenase; C048627:monolinolein; D010100:Oxygen