Novel insights into cyclooxygenases, linoleate diol synthases, and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs

Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O2. Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n − 6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n − 6 and n − 9 fatty acids and by analysis of D-KIE. COX-1 oxidized C20 and C18 fatty acids in the following order of rates: 20:2n − 6 > 20:1n − 6 > 20:3n − 9 > 20:1n − 9 and 18:3n − 3 ≥ 18:2n − 6 > 18:1n − 6. 18:2n − 6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n − 6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n − 6, but the 9E,12Z isomer was mainly subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-2H]18:2n − 6 with similar D-KIE (~ 53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n − 6 by COX-1 and COX-2 took place with a D-KIE of 3–5 as probed by incubations of [11,11-2H2]- and [11S-2H]18:2n − 6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n − 6 by COX-1 and 18:1n − 9 by 8R-DOX were both accompanied by large D-KIE (> 20).

► Deuterium kinetic isotope effect (D-KIE) larger than 20 can be explained by hydrogen tunneling.
► Fe- and Mn-LOX oxidize bis-allylic carbons with large D-KIE (> 50) and COX-1 with small (D-KIE < 4).
► COX-1 and 8R-DOX oxidize allylic carbons efficiently, but with large D-KIE (> 20).
► Hydrogen tunneling contributes to reaction rates of energetically demanding enzymatic reactions.