Product specificity of SBLOX-1 with natural PUFAs
Beside LA and AA being used widely as substrates of LOXs, other PUFAs such asa-
andc-linolenic acids, EPA and DHA have been studied. For example, using the
‘under-pressure lipoxygenation’ methodology (see Section 2.2.3), the n-6(S)-
HPODs ofa-linolenic and arachidonic acids have been synthesized (Table 1) in
near-quantitative manner on a 3 mM scale (0.1 M substrate concentration) using
a commercial preparation of SBLOX-1 (Martini et al., 1996a, Martini and Iaca-
zio, 1997).
Porter et al. (1979) have also shown usingc-linolenic acid (3.29 mM) that the
corresponding n-6 HPOD is formed exclusively with SBLOX-1 as catalyst (pH
9, 0 8 C). When EPA or DHA are used as substrates of SBLOX-1 the same n-6 spe-
cificity is observed, but only at high pH (Takagi et al., 1987). Indeed, with these two
substrates as well as with AA, pH-dependent double dioxygenations are observed. In
1977, Bildet al. were the first to report, using AA, such double dioxygenation cat-
alyzed by SBLOX-1 (Bild et al., 1977b). When the reaction was conducted at high
enzyme concentration and at pH 6.8 it was observed that two equivalents of dioxygen
were consumed per substrate equivalent, and that the initially formed conjugated
dienic monohydroperoxide (UV absorbance maximum at 238 nm) disappeared
slowly, leading to a conjugated trienic chromophore (UV absorbance maximum
at 260, 269, and 279 nm). The structure of the dihydroperoxide formed was
8,15-dihydroperoxy-(5Z,9E,11Z,13E)-eicosatetraenoic acid (8,15-diHPETE).
346 16 Properties and Applications of Lipoxygenases
Table 1.High substrate concentration ‘under-pressure lipoxygenation’ ofa-linolenic and arachidonic
acids. (From Martini et al., 1996a; Martini and Iacazio, 1997.)
Substrate Time (min) Yield (%) Product Enantiomeric
excess (%ee)
(Abs. conf.)
LnA 10 > 99 >99 (S)
AA 15 99 99 (S)
Figure 8. Double dioxygenation catalyzed by SBLOX-1 (Bild et al., 1977b; Van Os et al., 1981).