Front Matter

15.3.2 Specificity of mammalian LOXs

In contrast to plant cells, most mammalian tissues contain large amounts of AA and

in various animal cells–such as polymorphonuclear leukocytes, monocytes or

macrophages–this fatty acid is even the major polyenoic fatty acid. Since AA is

the major substrate for the formation of prostaglandins and leukotrienes, research

has been focused in the past on the oxidative metabolism of this fatty acid. How-

ever, in several mammalian cells and tissues and also in extracellular lipids (e.g.,

plasma lipoproteins), LA is more abundant than AA. Thus, the oxidative metabo-

lism of LA via the LOX pathway may also lead to bioactive compounds, which

may have been underestimated so far. In fact, (13S,9Z,11E)-13-hydroxy-9,11-octa-

decadienoic acid, the major oxygenation product of LAvia the 15-LOX pathway has

been shown to exhibit interesting biological activities (Ku ̈hn, 1996). Nevertheless,

for the time being AA metabolism remains in the center of eicosanoid research,

although investigations of the metabolic fate of other polyunsaturated fatty acids

may have become increasingly important during the past few years.

According to the currently used nomenclature, mammalian LOXs are categorized

with respect to their positional specificity of AA oxygenation into 5-LOXs, 8-LOXs,

12-LOXs, and 15-LOXs (Funk, 1996; Brash, 1999; Ku ̈hn and Thiele, 1999).

Although this nomenclature is straightforward and commonly accepted, it suffers

from several disadvantages, which may lead to confusion among scientist not work-

ing in the field. The major disadvantage of this nomenclature is that it is based on a

single enzyme property and does not consider other structural and functional enzyme

characteristics. Moreover, the positional specificity of LOXs is not an absolute en-

zyme property but depends strongly on the way that the enzyme interacts with the

substrate. This interaction is of course influenced by a variety of factors such as

substrate concentration (Ku ̈hn et al., 1990a), the physico-chemical state of the sub-

strate (Began et al., 1999), pH (Gardner, 1989) or temperature, but may also depend

on the structures of both, enzyme and substrate. If this hypothesis is correct, it may be

possible to alter the positional specificity by targeted modification of the substrate

and/or by site-directed mutagenesis of critical amino acids involved in positioning

the fatty acid substrate at the active site.

Alteration of positional specificity with C 20 fatty acid substrates of

mammalian and plant LOXs

Since site-directed mutagenesis requires detailed sequence information on various

LOX isoforms, the problem of enzyme/substrate interaction was initially approached

by targeted substrate modification. In 1967, Hamberg and Samuelsson investigated

the structural reasons for the positional specificity of the soybean LOX reaction

using different polyenoic fatty acids. They concluded that the distance of the bis-

allylic methylene where hydrogen abstraction takes place from the methyl end of

the substrate molecule appeared to be important (Hamberg and Samuelsson,

1967). Similar results were later on obtained with the rabbit 15-LOX (Ku ̈hn et

al., 1990b). From these data a topological model of enzyme substrate interaction

was developed suggesting that polyenoic fatty acids may penetrate the active site

15.3 The structural bases of the positional specificity of LOXs 321