Submission Date

7-22-2016

Document Type

Paper- Restricted to Campus Access

Department

Chemistry

Faculty Mentor

Amanda Reig

Comments

Presented during the 18th Annual Summer Fellows Symposium, July 22, 2016 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 meleninization and sclerotization processes. Understanding the kinetics of the polyphenol oxidation activity in these enzymes is crucial in determining the nature of the enzyme-substrate complex and the design of efficient target drugs and inhibitors. The de novo designed Due Ferri Single Chain (DFsc) proteins are water-soluble monomeric four-helix bundles that can readily bind metal cofactors, optimal for rapid kinetic studies of metal enzymes. Several variants on this scaffold, namely mutants of G4DFsc and 3-HisG4DFsc, have been shown to mimic diiron oxidase activity. Dicopper binding has been suggested in mutants such as E11H/E44H-G4DFsc and E11H-3HisG4DFsc, which contain high numbers of His residues in their active sites similar to what is found in nature dicopper oxidases. In this study, copper binding was confirmed by circular dichroism spectroscopy and catalytic oxidase active for copper-bound DFsc variants was determined by UV-Visible spectrophotometric assays. Specifically, the formation of a o-quinone coupling product with 3-methyl-2-benzothiazolinone hydrazone (MBTH), and oxidation of NADH in the presence of the copper-protein complex and a catechol derivative at a rate faster than that of aqueous copper(II) is indicative of catechol oxidase–like behavior. Catalytic oxidation was further supported by a change in activity when under anaerobic and reductive conditions.

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