Enzyme Genomics

at the University of Toronto

In all sequenced genomes, a large fraction of predicted genes (up to 50% in some genomes) encodes proteins of unknown biochemical function. Many global strategies are already being used to infer function (protein-protein interaction, gene expression analysis, structural studies).

As an additional approach to discover protein function, we have developed a series of general enzymatic assays with which to test purified unknown proteins for catalytic activity. These assays are designed to be of broad substrate specificity to enable the identification of the sub-class or sub-subclass of enzymes (esterases, phosphatases, proteases, nucleases, dehydrogenases) to which the specific protein belongs.

Proteins that have detectable activity are further characterized using more specific assays. Specifically: phosphatases are screened for activity against a panel of 93 physiological substrates, phosphodiesterases/nucleases – with 26 substrates, and esterases/lipases – with 37 substrates. The spectrophotometric assays are developed for 96-well plates, can be performed quickly and require only a few micrograms of protein.

Using these general assays, we have detected catalytic activity in over 200 purified proteins from various organisms produced by our structural proteomics group or our collaborators. Most of them are phosphatases, phosphodiesterases and esterases.

To characterize their cellular role, we are using additional approaches (gene knock-outs etc.) and are interested in collaboration with established labs. As well, we invite all researchers wishing to check their proteins for catalytic activity to send them to us for screening (conditions and terms of collaboration).

Last updated September 2011
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