binding pocket and is positioned near C 4 of the scissile fatty acid chain. Replacing it
by the hydrophilic threonine decreases activity 5- to 10-fold. Replacing F111, which
is near to C 6 , by tryptophan increases the relative specificity for short-chain tri-
glycerides. L208 and F94 line the hydrophobic crevice near C 10 and C 12. Replacing
them by bulkier and more hydrophilic residues decreases activity and increases re-
lative specificity for fatty acids of short and medium chain length. F213 is located at
the end of the hydrophobic crevice near C 16. Replacing it by the more bulky and
hydrophilic tyrosine reduces relative specificity for oleic and stearic acid. As a strat-
egy to predict mutants with specificity for chain lengths below a given cut-off, bind-
ing of longer chains can be inhibited by increasing size and polarity of a residue
positioned at thex-end of the longest chain to be accepted. Binding of substrates
of different chain length might also influence flexibility of the lipase, as it has been
concluded from molecular dynamics simulations of RML with and without inhibitor
(Peters et al., 1997). Opening of the lid and binding of fatty acid analogs substantially
reduced fluctuations of solvent-exposed loops, thus making the lipase more rigid.
90 5 Molecular Basis of Specificity and Stereoselectivity of Microbial Lipases
Figure 2. Two perpendicular schematic views of theRhizomucorlipase binding site, and a modeled
scissile fatty acid chain of length C 18 (Pleiss et al., 1998). The narrow bottom of the binding pocket and a
hydrophobic patch are shaded dark and light gray, respectively, as calculated by GRID (Goodford, 1985).
The position of five residues (see Table 1) which have been shown to mediate chain length specificity in
the homologous RDL and ROL are marked as dots.