Front Matter

AA with acceptable yields (Reddanna et al., 1990). This enzyme does not require

essential co-factors, and is stable over months in suspension without a significant

loss in activity when stored at –20 8 C (Reddanna et al., 1990). Unfortunately,

the positional specificity of this enzyme, as that of other plant LOXs (Feussner

and Ku ̈hn, 1995), is less stringent than that of the human enzyme. In fact, it converts

AA to a mixture of 11-, 8- and 5-HETE in a ratio of 1:1:2 (Figure 10, wild-type).

Since the amino acid sequence of the potato tuber LOX is available (Geerts et al.,

1994), it should be possible to alter the positional specificity by site-directed mu-

tagenesis to make the product pattern more specific. As one example of our approach

we created the V576F mutant of this enzyme (V576 aligns with F353 of the rabbit 15-

LOX) and obtained a highly specific product pattern of AA oxygenation (Figure 10).

Moreover, this mutation did not affect other biochemical parameters of the enzyme.

15.3.3 Alteration of positional specificity by modifying

the physico-chemical state of LOX substrates

The positional specificity of LOXs is not an absolute enzyme property, but depends

heavily on how the enzyme interacts with the substrate. This interaction is influenced

by a variety of factors such as substrate concentration (Ku ̈hn et al., 1990a), the phy-

sico-chemical state of the substrate (Began et al., 1999), pH (Gardner, 1989) or

temperature (Georgalaki et al., 1998), but may also depend on the structures of

both, enzyme and substrate. Since LOX substrates have a limited water solubi-

lity, aqueous preparations constitute a complex mixture of monomers, acidic

soap dimers and higher molecular structures such as uni- and/or multilaminar mi-

celles or liposomes. In addition, ionic and/or nonionic detergents are frequently used

for LOX assays to increase the availability of the substrate, and these detergents even

increase the complexity of the substrate suspension (Lopez-Nicolas et al., 1994;

1997a,b; Began et al., 1999). Under physiological conditions, polyenoic fatty acids

are bound predominantly to membranes (Glickman and Klinman, 1995), and this

association may also impact the reaction characteristics.

It has been shown recently that it is possible to change the positional specificity of

the soybean LOX-1-catalyzed reaction by altering the physico-chemical state of the

fatty acid substrate (Began et al., 1999). Therefore, polyenoic fatty acids were in-

serted into phosphatidylcholine micelles with their tail groups buried inside, and the

authors showed that these modified fatty acids were better substrates for soybean

LOX-1. With Tween 20-solubilized LA the enzyme had an alkaline pH optimum

and it exclusively formed the (13S)-hydroperoxy derivative (Gardner, 1989). How-

ever, with LA or AA inserted into phosphatidylcholine micelles, LOX-1 synthesized

exclusively the (9S)-hydroperoxy- or (5S)-hydroperoxy derivative, respectively, and

this transformation was no longer dependent on the pH value. Thus, LOX-1, could

utilize polyenoic fatty acids bound to membranes as physiological substrates, and it

utilized the carboxylic head group of the fatty acid inserted in the phosphatidylcho-

line micelles as a recognition site for 9-lipoxygenation. This was confirmed by ac-

tivity measurements using the fatty acid methyl esters as substrates.

330 15 Application of Lipoxygenases and Related Enzymes