Interchange of catalytic activity within the 2-enoyl-coenzyme a hydratase isomerase superfamily based on a common active site template

The structures and chemical pathways associated with the members of the 2-e noyl-CoA hydratase/isomerase enzyme superfamily are compared to show that a common active site design provides the members of this family with a CoA b inding site, an expandable acyl binding pocket, an oxyanion hole for bindin g/polarizing the thioester C=O, and multiple active site stations for the p ositioning of acidic and basic amino acid side chains for use in proton shu ttling. It is hypothesized that this active sire template can be tailored t o catalyze a wide range of chemical transformations through strategic posit ioning of acid/base residues among the active site stations. To test this h ypothesis, the active site of one member of the 2-enoyl-CoA hydratase/isome rase 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 hydra tion of crotonyl-CoA, absent in the wild-type 4-chlorobenzoyl-CoA dehalogen ase, was shown to occur with the structurally modified 4-chlorobenzoyl-CoA dehalogenase at k(cat) = 0.06 s(-1) and K-m = 50 mu M.