Front Matter

are aligned at the active site, and whether the rules established for polyenoic fatty

acids are also applicable for complex substrates.

Conversion of the human arachidonate 5-LOX to 8- and 15-lipoxygenating

species

As indicated earlier, it is relatively easy to convert a 15-LOX to a 5-lipoxygenating

species when the structure of the LOX substrate is altered appropriately. In contrast,

previous attempts failed to transform a 12/15-LOXs to a 5-lipoxygenating enzyme by

site-directed mutagenesis. When the residues 418 and 419 of the human and the

rabbit reticulocyte-type 15-LOX were mutated to the amino acids present in the

human 5-LOX, inactive enzyme species resulted.

As indicated before, two theories exist which relate the positional specificity of 5-

LOXs to that of 12/15-lipoxygenating enzymes:

1. The space-related theory (Funk and Loll, 1997; Skrzypczak-Jankun et al., 1997;

Browner et al., 1998), which suggests that the product specificity of a LOX is

determined by the volume of the substrate-binding pocket. Fatty acids are bound

at the active site of all LOXs in a conserved AA-like orientation. Arachidonate

12/15-LOXs have a smaller substrate-binding cleft as 5-LOXs and thus may not

penetrate as deep into the pocket, as may be the case for 5-LOX. On the other

hand, the space available at the active site of 5-LOXs may allow a substrate

alignment optimal for 5-lipoxygenation.

2. The orientation-related theory (Ku ̈hn et al., 1986; Lehmann, 1994; Funk and Loll,

1997; Prigge et al., 1998): according to this hypothesis 5-lipoxygenation requires

an inverse head-to-tail orientation of the substrate so that fatty acid substrates

may slide into the binding pocket with the carboxylic group ahead. With this

hypothesis the stereochemistry of (5S)-lipoxygenation can easily be ex-

plained. An inverse substrate orientation would be energetically favored, if polar

or even charged amino acids were present at the bottom of the substrate-binding

pocket. However, molecular modeling of the enzyme/substrate interaction sug-

gested that the closest potential positive charge in the human 5-LOX would be

H354 which is located at a distance of more than 6 A ̊from the position of the

modeled substrate carboxylate group (Browner et al., 1998).

Although there is a substantial body of circumstantial experimental evidence favor-

ing the orientation-related hypothesis, neither of the two theories has been proven

experimentally. The orientation hypothesis can only be proven if co-crystallization

studies of a 5-LOX with a substrate fatty acid are carried out. The space-hypothesis

can be tested by site-directed mutagenesis. As mentioned above, at least all attempts

failed to convert a mammalian 15-LOX to a 5-lipoxygenating species. We recently

approached this problem with an inverse strategy, attempting to convert the human

leukocyte 5-LOX to a 15-lipoxygenating enzyme. In order to decrease the volume of

the substrate-binding cage, we mutated the sequence determinants of the 5-LOX to

the more space-filling counterparts present at these positions in 15-LOXs. An A424I

15.3 The structural bases of the positional specificity of LOXs 327