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Hye-Kyung Kim, Jian-Wei Liu, Paul D. Carr and David L. Ollis
Research School of Chemistry, Australian National University,
Building 35, Science road, ACT 0200, Australia
The enzyme dienelactone hydrolase (DLH) has undergone directed evolution to produce a
series of mutant proteins that have enhanced activity towards the non-physiological
substrates, -naphthyl acetate and p-nitrophenyl acetate.
In terms of steady state kinetics, the mutations caused a drop in the Km for the hydrolysis
reaction with these two substrates. For the best mutant, there was a 5.6 fold increase in
kcat/Km for the hydrolysis of -naphthyl acetate and a 3.5 fold increase was observed for pnitrophenyl acetate. For -naphthyl acetate the pre-steady state kinetics revealed that the
rate constant for the formation of the covalent intermediate had increased. The mutations
responsible for the rate enhancements map to the active site.
The structures of the starting and mutated proteins revealed small changes in the protein
due to the mutations, while the structures of the same proteins with an inhibitor (PMSF)
co-crystallized in the active sites indicated that the mutations caused significant changes in
the way the mutated proteins recognized the substrates. Within the active site of the mutant
proteins, the inhibitor was rotated by about 180 with respect to the orientation found in the
starting enzyme. This rotation of the inhibitor caused the displacement of a large section of
a loop on one side of the active site. Residues that could stabilize the transition state for the
reaction were identified.