2023 101 P

Successful Stabilization of an Enzyme Kinetic Assay for a Novel aAetyltransferase from Pseudomonas aeruginosa is Guanidine HCl Concentration Dependent

By: Pamela Caro De Silva

Department: Chemistry & Biochemistry

Faculty Advisor: Dr. Misty Kuhn

In general, discontinuous kinetic assays use a type of secondary reaction to detect a product. Often, this requires a denaturant which is used to stop the enzymatic reaction. Then a molecule that can be observed colorimetrically is added to the solution to quantify the amount of product produced during a reaction. One common denaturant that we use to stop in our enzyme reactions is guanidine chloride (GdnHCl), and we use DTNB (Ellman’s reagent) to react with the sulfhydryl of the CoA products. The GdnHCl solution works by unfolding the protein to stop the reaction so it can no longer catalyze the reaction. Then CoA products generated during the reaction will react with DTNB to form the anion TNB2- which is measured at A412 nm with a spectrophotometer. While we were trying to identify a substrate for a novel acetyltransferase, we observed precipitation in the reaction. During the course of troubleshooting the assay, we were able to isolate the causative agent. Additionally, we showed that contrary to previous primary literature, the siderophore Ferrichrome, was not a substrate for this enzyme. These results show a significant change in our assay is required for some acetyltransferases and serves as a cautionary tale for others who also utilize a similar approach.