Differential characteristics of human 15-lipoxygenase isozymes and a novelsplice variant of 15S-lipoxygenase

The lipoxygenases (LOs) are a family of nonheme iron dioxygenases that cata lyse the insertion of molecular oxygen into polyunsaturated fatty acids. Fi ve members of this gene family have been described in man, 5-LO, 12S-LO, 12 R-LO, 15-LO and 15S-LO. Using partially purified recombinant 15S-LO enzyme and cells constitutively expressing this protein, we have compared the acti vity, substrate specificity, kinetic characteristics and regulation of this enzyme to that previously reported for 15-LO. 15S-LO has a threefold highe r K-m, similar V-max and increased specificity of oxygenation for arachidon ic acid, and a similar K-m but decreased V-max for linoleic acid in compari son to 15-LO. Unlike 15-LO, 15S-LO is not suicide inactivated by the produc ts of fatty acid oxygenation. However, in common with other LOs, 15S-LO act ivity is regulated through calcium-dependent association of the enzyme with the membrane fraction of cells. In addition, whilst independently cloning the recently described 15S-LO, we identified a splice variant containing an in-frame 87-bp deletion correspo nding to amino acids 401-429 inclusive. Modelling of the 15S-LO and subsequ ent studies with partially purified recombinant protein suggest that the de leted region comprises a complete alpha-helix flanking the active site of t he enzyme resulting in decreased specificity of oxygenation and affinity fo r fatty acid substrates. Alternative splicing of 15S-LO would therefore provide a further level of r egulation of fatty acid metabolism. These results demonstrate that there ar e substantial differences in the enzyme characteristics and regulation of t he 15-LO isozymes which may reflect differing roles for the proteins in viv o.