Front Matter

15.3 The structural bases of the positional specificity of

LOXs and alteration of the positional specificity by

modifying the substrate or by site-directed

mutagenesis

The positional specificity of LOXs is a result of two more or less independent cat-

alytic processes.

1. Regio- and stereospecific hydrogen removal: with substrate fatty acids containing

several doubly allylic methylenes such as linolenic acid (LeA), AA or eicosapen-

taenoic acid hydrogen abstraction from two, three or four doubly allylic methy-

lenes, respectively, is possible. The positional specificity of the enzyme deter-

mines which of these bisallylic methylenes will be attacked.

2. Regio- and stereospecific oxygen insertion: when hydrogen is abstracted from a

certain doubly allylic methylene, molecular oxygen can be introduced either at

the [+2] or at the [-2] position (Figure 2). Thus, a fatty acid containing three

doubly allylic methylenes such as AA can be oxygenated by a LOX to 6 re-

gio-isomeric hydroperoxy derivatives (HPETEs), namely 15- and 11-HPETE

(originating from C 13 hydrogen removal), 12- and 8-HPETE (C 10 hydrogen re-

moval) and 9- and 5-HPETE (C 7 hydrogen removal).

In the early days of LOX research, before structural data on this enzyme family

became available, the mechanistic reasons for the positional specificity were inves-

tigated by targeted modification of LOX substrates. Experiments with AA isomers

(positional isomerism of the double bonds) suggested that the positional specificity

of LOXs may not be an absolute enzyme property, but may depend on the orientation

of the substrate molecule at the active site of the enzyme (Hamberg and Samuelsson,

1967; Ku ̈hn et al., 1990b). The experimental data indicated that, for oxygenation of

polyenoic fatty acids by the LOXs from rabbit reticulocytes and soybeans, the dis-

tance of the bisallylic methylene from the methyl end of the fatty acid is important.

Furthermore, it was concluded that arachidonate 12-LOXs may have a deeper sub-

strate-binding pocket than 15-LOXs which makes an optimal substrate orientation

for 12-lipoxygenation more likely (Figure 3A, shown for LA).

After sequence data of various LOXs became available, the mechanistic reasons

for the positional specificity were explored by targeted alteration of the enzymes.

Multiple sequence alignments of various LOXs with different positional specifici-

ties suggested critical amino acids in mammalian 12- and 15-LOXs which may be

considered as primary determinants of the positional specificity (Sloane et al., 1991;

Borngra ̈ber et al., 1996). When the space-filling M419 or F353 of the human and/or

rabbit reticulocyte-type 15-LOXs are mutated to smaller residues, the substrate fatty

acids may slide in farther into the substrate-binding pocket which favored arachi-

donate 12-lipoxygenation (Sloane et al., 1991; Borngra ̈ber et al., 1996). When

the substrate binds deeper, the bisallylic methylene at C 10 of AA approaches the

catalytically active nonheme iron, whereas C 13 is dislocated. In these experiments

the site of hydrogen abstraction was altered by site-directed mutagenesis, but the

direction of the [+2] radical rearrangement remained unchanged.

15.3 The structural bases of the positional specificity of LOXs 313