Methods for discovering catalytic activities for pseudokinases

Pseudoenzymes resemble active enzymes, but lack key catalytic residues believed to be required for activity. Many pseudoenzymes appear to be inactive in conventional enzyme assays. However, an alternative explanation for their apparent lack of activity is that pseudoenzymes are being assayed for the wrong reaction. We have discovered several new protein kinase-like families which have revealed how different binding orientations of adenosine triphosphate (ATP) and active site residue migration can generate a novel reaction from a common kinase scaffold. These results have exposed the catalytic versatility of the protein kinase fold and suggest that atypical kinases and pseudokinases should be analyzed for alternative transferase activities. In this chapter, we discuss a general approach for bioinformatically identifying divergent or atypical members of an enzyme superfamily, then present an experimental approach to characterize their catalytic activity.