[23] Use of phage display and transition-state analogs to select enzyme variants with altered catalytic properties: Glutathione transferase as an example

Evidence suggests that naturally occurring proteins adopt a limited number of folds, whereas the variety of primary structures is essentially unlimited. It can therefore be concluded that most naturally evolved proteins have arisen by redesign of previously existing protein scaffolds. This approach to protein design appears particularly obvious in protein families where structural similarities are pronounced even though functional properties are highly divergent. The soluble glutathione transferases (GSTs) are a family of detoxication enzymes with diverse substrate specificities. The multiple forms of GSTs have been divided into classes, and as a first approximation, functional properties have been found to be correlated with the amino acid sequences. Even though the overall identities in the amino acid sequences are limited among the known GSTs, the protein folds of the different classes are similar. This finding agrees with the proposal that the GSTs have undergone divergent evolution from a common ancestral protein. The evolutionary perspective thus suggests that GSTs have stable structures, which by combinatorial rearrangements can be redesigned for novel functions. This chapter describes the use of transition-state analogs for the selection of GSTs with novel catalytic properties from libraries of active site mutants. The GST variants are expressed in functional form on filamentous phage and variant GSTs with proper affinities are isolated by affinity adsorption, using transition-state analogs as ligands.