Candida antarcticalipase B (CALB) is a member of the funnel-like lipases (Pleiss
et al., 1998). Its binding pocket is an elliptical, narrow funnel of 9.54.5 A ̊(see
Figure 1). Up to C 4 , the scissile fatty acid binds to a cleft at the hydrophilic bottom of
the funnel, which is formed by D134 and the catalytic S105. It is lined by T138, I189,
and V190 on its left-hand side, by Q157 and the oxyanion hole residue T40 on its
right-hand side. At the end of this cleft, near Q157, the fatty acid kinks sharply and
follows the left-hand wall. Up to C 7 , the binding site is a narrow cleft, from C 7 to C 13
it becomes smooth and hydrophobic. This hydrophobic binding site is formed by
V154, I285, L144 and V149. Its width decreases with the distance from the active
site with its minimum at C 13 of the fatty acid chain. Here, at 13.5 A ̊above the bottom,
it reaches the end of the funnel. Compared to the long, well-defined hydrophobic
crevice of RML, the scissile fatty acid binding site of CALB is relatively short
(C 13 ) and has a small hydrophobic area located at the wall of the binding funnel.
Based on shape and physico-chemical properties of the binding pocket, chain length
preference of CALB is expected to be shifted towards shorter chains compared to
RML. Under experimental conditions, where the scissile fatty acid binding site is the
primary determinant for chain length specificity (Rangheard et al., 1992), CALB has
indeed high activity for short and medium chain length fatty acids and decreasing
activity for long-chain fatty acids (Figure 3), while RML has relatively low activity
for short-chain fatty acids, but increasing activity for longer-chain fatty acids (Rang-
heard et al., 1992; Kirk et al., 1992). If, however, assays are used which involve
activity at a water-substrate interface, chain length dependency may change drasti-
cally, as it has been shown for the hydrolysis of triglycerides by RML (Berger and
Schneider, 1991). In addition to the scissile fatty acid binding site, other structural
elements will play a role in mediating chain length specificity, such as the alcohol
binding site and the lid. W89 inHumicola lanuginosalipase is part of the lid and does
not contribute to the scissile fatty acid binding site. However, mutating W89 resulted
in a change of the chain length profile (Martinelle et al., 1996), which has been
assigned to effects on binding the acyl chain of the substrate.
5.3 Modeling and engineering of fatty acid specificity 91
Figure 3. Relative specificity constantskcat/Kmof RML and CALB (normalized to C 6 ) for fatty acid
chains of varying length (Pleiss et al., 1998); RML: alcoholysis of monoester byn-propanol (Rangheard et
al., 1992) and esterification ofn-octanol in hexane (Kirk et al., 1992); CALB: esterification ofn-octanol in
hexane (Kirk et al., 1992) and esterification of ethylD-glucopyranoside (Kirk et al., 1992).