CONVERSION OF HUMAN 15-LIPOXYGENASE TO AN EFFICIENT 12-LIPOXYGENASE - THE SIDE-CHAIN GEOMETRY OF AMINO-ACID-417 AND AMINO-ACID418 DETERMINE POSITIONAL SPECIFICITY

Positional specificity determinants of human 15-lipoxygenase were examined by site-directed mutagenesis and by kinetic analysis of the wild-type and variant enzymes, By comparing conserved differences among sequences of 12-and 15-lipoxygenases, a small region responsible for functional differences between 12- and 15-lipoxygenases has been identified, Furthermore, the replacement of only two amino acids in 15-lipoxygenase (at 417 and 418 in the primary sequence) by those found in certain 12-lipoxygenases results in an enzyme that has activity similar to 12-lipoxygenase. An examination of the activity of nine variants of lipoxygenase demonstrated that the amino acid side-chain bulk and geometry of residues 417 and 418 are the key components of the positional specificity determinant of 15-lipoxygenase. Overexpression of a variant (containing valines at positions 417 and 418) that performs predominantly 12-lipoxygenation was achieved in a baculovirus-insect cell culture system, This variant was purified to >90% homogeneity and its kinetics were compared with the wild-type 15-lipoxygenase. The variant enzyme has no change in its apparent K-M for arachidonic acid and a minor (3-fold) change in its V-max. For linoleic acid, the variant has no change in its K-M and a 10-fold reduction in its V-max, as expected for an enzyme performing predominantly 12-lipoxygenation. The results are consistent,vith a model in which two amino acids of 15-lipoxygenase (isoleucine 417 and methionine 418) constitute a structural element which contributes to the regiospecificity of the enzyme, Replacement of these amino acids with those found in certain 12-lipoxygenases results in an enzyme which can bind arachidonic acid in a catalytic register that prefers 12-lipoxygenation.