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pubmed:abstractText |
The structures and chemical pathways associated with the members of the 2-enoyl-CoA hydratase/isomerase enzyme superfamily are compared to show that a common active site design provides the members of this family with a CoA binding site, an expandable acyl binding pocket, an oxyanion hole for binding/polarizing the thioester C=O, and multiple active site stations for the positioning of acidic and basic amino acid side chains for use in proton shuttling. It is hypothesized that this active site template can be tailored to catalyze a wide range of chemical transformations through strategic positioning of acid/base residues among the active site stations. To test this hypothesis, the active site of one member of the 2-enoyl-CoA hydratase/isomerase family, 4-chlorobenzoyl-CoA dehalogenase, was altered by site-directed mutagenesis to include the two glutamate residues functioning in acid/base catalysis in a second family member, crotonase. Catalysis of the syn hydration of crotonyl-CoA, absent in the wild-type 4-chlorobenzoyl-CoA dehalogenase, was shown to occur with the structurally modified 4-chlorobenzoyl-CoA dehalogenase at kcat = 0.06 s-1 and Km = 50 microM.
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