Type of Proposal
Faculty of Science
Dr. Bulent Mutus
24-3-2015 2:00 PM
24-3-2015 2:50 PM
Importance of the Project
My research is to follow-up on the findings of a previous student which proposed the presence of a potentially essential conserved lysine residue neighbouring the redox active sites of both a and a' domains of protein disulfide isomerase (PDI). I will be employing various techniques to further study the role of these active site lysine residues (Lys57 and Lys401 of human PDI, specifically).
Existing State of Knowledge
PDI has two redox-active thioredoxin-like domains: a and a', each with a highly conserved -CXXC- active site motif. The mechanistic workings of this motif are well characterized. It has been shown by a Japanese research group that the ability of PDI to catalyze the isomerization or oxidation-reduction of a substrate's disulfide bonds depends not only on the described motif, but also a neighbouring lysine residue found directly C-terminal to such.
Is an active site lysine residue, found quite conserved, of catalytic importance to PDI's oxidoreductase activity?
Fluorogenic assay to measure enzyme kinetic data.
Ref: Raturi, A., and Mutus, B., 2007, "Characterization of redox state and reductase activity of protein disulfide isomerase under different redox environments using a sensitive fluorescent assay," Free Radical Biol. & Med., v. 43, p. 62-70.
pH-dependency kinetic assays.
Computational molecular dynamics to further study structural aspects of PDI (collaborative work).
Active site lysine mutations have proven detrimental to the catalytic activity of PDI as indicated by appreciable decreases in both both Vmax and catalytic efficiency. Consistent kM values between the wild type and mutant enzymes give indications that the active site conformation is not altered as a result of mutation. Computational analysis should indicate the primary role of active site lysine residues in PDI's oxidoreductase activity.
Investigating the Impact of Active Site Mutations on the Oxidoreductase Activity of Protein Disulfide Isomerase