Novel Yeast Protease

Peter Lyons (Biology)

Purification and characterization of a novel yeast protease

The biochemical function of a peptidase is to cut up proteins. Sometimes this cutting occurs at very specific locations within the protein in order to form a mature protein or to regulate its activity in some way. Sometimes, however, this cutting occurs in many parts of the protein, in order to break the protein down into smaller pieces, and ultimately into individual amino acids of which a protein is made. These digestive enzymes are commonly found within the intestinal tracts of organisms, or within the lysosome of animal cells and the vacuole of plant and fungal cells. One type of digestive enzyme is a metallocarboxypeptidase, which functions to trim individual amino acids from one end of a substrate protein.

The yeast Saccharomyces cerevisiae contains one of these metallocarboxypeptidases. This enzyme, Ecm14, appears to have a unique enzymatic mechanism, in which a lysine is used to activate the water molecule used to cut a peptide bond, rather than the typical glutamate as in all other metallocarboxypeptidases. In order to prove this unique mechanism, experiments must be done to purify the enzyme and characterize its activity using common techniques of enzymology. The goal of this project is to purify Ecm14 in soluble and active form. This will require the expression of Ecm14 at lower temperatures, for varying lengths of time, and along with over-expression of its probable activating protease, Pep4. If successful, this will provide necessary preliminary data for a larger grant application to the National Science Foundation.

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