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

Positional specificity determinants of human 15-lipoxygenase were exam ined by site-directed mutagenesis and by kinetic analysis of the wild- type and variant enzymes, By comparing conserved differences among seq uences of 12-and 15-lipoxygenases, a small region responsible for func tional differences between 12- and 15-lipoxygenases has been identifie d, Furthermore, the replacement of only two amino acids in 15-lipoxyge nase (at 417 and 418 in the primary sequence) by those found in certai n 12-lipoxygenases results in an enzyme that has activity similar to 1 2-lipoxygenase. An examination of the activity of nine variants of lip oxygenase demonstrated that the amino acid side-chain bulk and geometr y of residues 417 and 418 are the key components of the positional spe cificity determinant of 15-lipoxygenase. Overexpression of a variant ( containing valines at positions 417 and 418) that performs predominant ly 12-lipoxygenation was achieved in a baculovirus-insect cell culture system, This variant was purified to >90% homogeneity and its kinetic s 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 chan ge 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 co nsistent,vith a model in which two amino acids of 15-lipoxygenase (iso leucine 417 and methionine 418) constitute a structural element which contributes to the regiospecificity of the enzyme, Replacement of thes e 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.