of 12/15-LOXs with the methyl end (Ku ̈hn et al., 1986; Lehmann, 1994). According
to this model (Figure 7) AA is oxygenated at C 15 when the substrate is aligned in such
a way that the double allylic methylene C 13 is localized in close proximity to the
hydrogen acceptor (nonheme iron). In contrast, AA 12-oxygenation may result
when the substrate fatty acid penetrates somewhat deeper into the binding pocket
to approach the bisallylic C 10 to the iron (Figure 7). In other words, a shallow sub-
strate-binding pocket may favor AA 15-oxygenation, whereas a deeper cleft may
lead to 12-HETE formation. If the depth of the binding cleft is in between, an en-
zyme with a dual positional specificity results (Figure 7).
In order to obtain additional evidence indicating the importance of substrate struc-
ture for the positional specificity, the oxygenation of 15-HETE by the LOXs from
soybeans (van Os et al., 1981) and rabbits (Schwarz et al., 1998) was investigated.
This substrate was converted by the rabbit enzyme mainly to (14R,15S)-DiH(P)ETE.
In contrast, the soybean enzyme formed a mixture of (5S,15S)- and (8S,15S)-
DiH(P)ETE. Most interestingly, after methylation the rabbit LOX mainly catalyzed
5-lipoxygenation, and the share of (5S,15S)-DiH(P)ETE formed by the soybean en-
zyme was strongly increased (Schwarz et al., 1998). These data indicated that with
15-HETE as substrate the arachidonate 15-LOXs from rabbit reticulocytes and soy-
beans constitute 5-LOXs. An inverse head to tail orientation of the substrate (Figure
8) may be discussed as a mechanistic reason for this unexpected product pattern, and
additional experimental evidence exists supporting this hypothesis:
* Methylation of 15-HETE strongly stimulated its oxygenation rate. This result may
be explained by the fact that methylation increases the hydrophobicity of the
carboxy terminus, reducing the energy barrier associated with burying the carbox-
ylate group in the hydrophobic substrate-binding pocket. In this context it is of
particular interest that the oxygenation rates of the substrates which are oxyge-
nated at C-15 (AA, 5-HETE, 8-HETE) were not augmented upon methylation, but
were even impaired (Schwarz et al., 1998).
322 15 Application of Lipoxygenases and Related Enzymes
Figure 7. Binding of polyenoic fatty acids at LOXs exhibiting a singular or dual positional specificity.
The solid circles represent the hydrogen acceptor of the enzyme (nonheme iron) and the ‘horse-shoe-like’
structure symbolizes the hydrophobic substrate-binding pocket of the enzyme. The estimated distance of
the hydrogen acceptor from the bottom of the hydrophobic pocket is given in methyl groups.