Angewandte
Chemie
vicinity of the active site that serves as a means to stabilize
hydrogen peroxide attack on a putative acyl-enzyme
intermediate (Figure 3). Modeling also shows that subtili-
sin, which also has a Ser–His–Asp catalytic triad but a
different protein fold, lacks perhydrolase activity likely
Figure 3. Proposed molecular basis of perhydrolase activity in an
esterase from P. fluorescens: the formation of the second tetrahedral
intermediate (after nucleophilic attack by the substrate peroxide) is
facilitated and subsequently stabilized by a key hydrogen bond in
the Leu29Pro mutant.
because the perhydrolysis tetrahedral intermediate lacks
key hydrogen bonds (Supporting Information). A similar
approach may be used to increase the perhydrolase activity
of other serine hydrolases or to alter the preferred
nucleophile to substrates other than water or hydrogen
peroxide.
Received: December 21, 2004
Published online: March 31, 2005
Keywords: enzyme catalysis · hydrolases · molecular
modeling · mutagenesis · peroxides
Figure 2. Models of the second tetrahedral intermediates of peracetic acid
formation: a) Leu29Pro PFE has increased perhydrolase activity and a
hydrogen bond between the backbone carbonyl oxygen atom of Trp28 and
.
À
À
the peroxide substrate hydroxy group (O O=2.7 ꢀ, O···H O
angle=1338); b) wild-type PFE has low perhydrolase activity and a weak
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À
À
hydrogen bond in the same location (O O=3.2 ꢀ, O···H O
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