1983), or from snake venom such asCrotalus atrox(Keith et al., 1981; Brunie et al.,
1985),Naja naja atra(Scott et al., 1990b) orAgkistrodon halyspallas (Wang et al.,
1996), are very similar. Some enzymes show a tendency to form dimers or higher
oligomers (Fremont et al., 1993). PLA 2 from bee venom, belonging to group III, has
little homology with the pancreatic and snake enzymes, but significant similarity in
the three-dimensional structure and in the catalytic mechanism (Scott et al., 1990a).
Several of the bee venom isoforms areN-glycosylated (Hollander et al., 1993), but
without significant influence on the kinetic properties of the enzyme (Dudler et al.,
1992).
The active sites of secretory PLA 2 s are composed of a hydrophobic channel with
an Asp-His catalytic dyad. The catalytic action of PLA 2 has probable similarities to
that of serine proteases, but does not proceed via an acyl enzyme intermediate.
Rather, it utilizes the His residue in the active site, assisted by an Asp residue, to
polarize a bound H 2 O, which then attacks the carbonyl group (Figure 4). The
Ca2+ion, located in the conserved Ca2+-binding loop, may stabilize the transition
state (Yu and Dennis, 1991a).
Much effort has been directed to mapping the interfacial binding site important for
the kinetic phenomenon of interfacial activation (see Section 1.4.2). For pancreatic
PLA 2 it has been proposed to be the flat external surface that surrounds the active-site
slot, including cationic and hydrophobic residues of theN-terminus, the C-terminus
and some other residues (Scott, 1997; Yuan and Tsai, 1999). By electron paramag-
netic resonance spectroscopy, Lin et al. (1998) defined the interfacial binding surface
of bee venom PLA 2 as a patch of hydrophobic residues. For PLA 2 from pig (Van den
Berg et al., 1995b) and beef (Yuan et al., 1999a), the structures in solution have also
been determined by multidimensional NMR spectroscopy, revealing flexibility of
several structural regions, e.g., theN-terminus, playing a role in interfacial activa-
tion.
Because of the great importance of interfacial activation of PLA 2 s an evaluation of
substrate specificity is very difficult (see Section 12.4.3). Absolute specificity, how-
ever, is found for thesn-2 position of the substrate, corresponding to the naturally
occurring enantiomeric form of phospholipids. The various secretory PLA 2 s show
somewhat different preferences with respect to the polar head groups (Rogers et al.,
1996), which can be modified by site-directed mutagenesis (Bhat et al., 1993; Bei-
boer et al., 1995), as well as with respect to the chain length (Lewis et al., 1990) and
the saturation degree (Bayburt et al., 1993) of the fatty acyl chains. Although PLA 2 is
226 12 Phospholipases Used in Lipid Transformations
Figure 4. Interactions of the catalytic diad D99–H48 with the substrate and Ca2+in secretory PLA 2 s.
(Mechanism adapted from to Yu and Dennis, 1991.)