Article
Biochemistry, Vol. 49, No. 37, 2010 7987
with the nerve agent analogues indicated that kcat/Km should be
enhanced over that of wild-type PTE, which was observed. The
H254G/H257W/L303T mutant exhibited a single phase during
the hydrolysis of racemic GD. This phase is dominated by the
hydrolysis of the two SP stereoisomers. The observed value of
kcat/Km was in rough agreement with that obtained for the
hydrolysis of the racemic GD by the wild-type enzyme, but this
mutant is significantly enhanced in the hydrolysis of the two most
toxic stereoisomers.
The reaction catalyzed by the H257Y/L303T mutant exhibited
two exponential phases during the time courses for the hydrolysis
of racemic GD, with the faster phase being the hydrolysis of the
two SP diastereomers and the slower phase being due to the
hydrolysis of the two RP diastereomers. The values of kcat/Km for
the hydrolysis of racemic GD were significantly higher than what
was predicted from the investigations with the nerve agent
analogues.
The use of the nerve agent analogues greatly simplifies the
identification of PTE variants with improved ability to hydrolyze
the more toxic stereoisomers of the authentic nerve agents GB
and GD. While the results with analogues and the specific nerve
agents are not identical, these results indicate that the findings
with analogues accurately predict improvements against GB and
GD. Comparison of the results obtained with the nerve agents
GB and GD and their respective analogues indicates that these
compounds may provide a more stringent test of the stereochem-
ical preferences of PTE based on the phosphoryl center. The
changes in kcat/Km obtained with the analogues are largely
reflected in the values of kcat/Km found with the authentic nerve
agents. These strategies have allowed the isolation of variants
that are significantly improved for the hydrolysis of both GB and
GD and with a stereochemical preference for the more toxic SP
stereoisomers.
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diisopropyl-fluorophosphatase from squid