simulations were conducted to identify alterations in amino acid sequence that might
impact substrate specificity. Appropriate mutants were then constructed by directed
mutagenesis. Strategies for altering chain length specificity included the introduction
of bulky hydrophobic residues at discrete positions in the acyl binding groove, the
creation of salt bridges by introducing polar or charged residues across the groove
from one another, and the replacement of hydrophobic amino acids with polar ones.
Studies with single mutants had identified two sites, at positions 112 and 209
(Figure 4), located roughly across the binding groove from one another and midway
down its length, where amino acid sequence changes affected substrate selectivity
(Joerger and Haas, 1994). In each of these mutants the introduced amino acid was a
tryptophan (Trp). In each case, mutation increased the relative activity toward short-
chain substrates relative to long-chain ones. Molecular dynamics simulations sug-
gested that the Trp might project across the acyl binding groove, preventing access
by fatty acids longer than about four carbons in length. It was postulated that if Trp
residues were introduced simultaneously at both sites within the same enzyme, geo-
metric or steric constraints might potentiate the effects of the individual mutations on
substrate selectivity. Such was subsequently found to be the case, with the double-
mutant lipase displaying a short-chain selectivity as much as 40 times greater than
4.7 Probing the role of structure-function relationships in substrate selectivity 81
Figure 4. Structural model of the active site region ofRhizopus delemarlipase, based on X-ray cry-
stallography and computer-assisted molecular modeling. In this view, thesn-3 fatty acyl side chain of
tricaprylin is shown (depicted as a dotted van der Waals surface), while the remainder of the triacylgly-
ceride is not displayed. The catalytic residues Ser145, Asp204, and His257 are labeled, along with Thr83,
which forms the oxyanion hole that stabilizes the catalytic intermediate. Selected residues lining the acyl
binding and catalytic sites are shown. Residues forming part of the acyl binding site that were targeted for
mutagenesis (Joerger and Haas, 1994; Klein et al., 1997a,b) are numbered. Modeling of the docking of
substrate into the active site was conducted by energy minimization and molecular dynamics simulation.