Submission Date

7-21-2017

Document Type

Paper- Restricted to Campus Access

Department

Chemistry

Faculty Mentor

Amanda Reig

Second Faculty Mentor

Ryan Walvoord

Comments

Presented during the 19th Annual Summer Fellows Symposium, July 21, 2017 at Ursinus College.

Supported by a National Institutes of Health Academic Research Enhancement Award (AREA) grant (R15-GM110657).

Project Description

Binuclear copper enzymes, such as tyrosinase and catechol oxidase, are involved in the melaninization and sclerotization processes. Understanding the mechanism of the diphenol oxidn. activity in these enzymes is crucial to design efficient target drugs and inhibitors. One way to gain mechanistic insight into these processes is through the design and characterization of protein-based model systems. The de novo designed Due Ferri Single Chain (DFsc) proteins are water-sol. monomeric four-helix bundles that can readily bind metal cofactors. Several variants on this scaffold, mutants of G4DFsc and 3HisG4DFsc, have been shown to mimic natural diiron oxidases. Interestingly, copper titrations have shown 2-, 3-, and 4-His G4DFsc variants bind two copper ions. There is also evidence that the Cu(I) form of these proteins can activate molecular oxygen. Catalytic oxidase activity for these DFsc variants was detd. using UV-Visible spectrophotometric assays. Specifically, the formation of an o-quinone coupling product with 3-methyl-2-benzothiazolinone hydrazone (MBTH) in the presence of the copper-protein complex and trihydroxybenzene (THB) is indicative of catechol oxidase-like behavior. These results are supported by Michealis-Menten kinetics studies. Catalytic oxidn. was further supported by changes in activity when the assays were conducted under anaerobic and reductive conditions, and in response to active site variations. Further development is focused towards the mutagenesis of a biomimetic 6-His variant, and turn-on fluorescent probes for sensitive kinetics.

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