Honors Theses

Date of Award

Spring 5-14-2023

Document Type

Undergraduate Thesis


Chemistry and Biochemistry

First Advisor

Saumen Chakraborty

Second Advisor

Jonah Jurss

Third Advisor

Kensha Clark

Relational Format



The design of artificial enzymes has been a topic of significant interest in the field of biochemistry, as they can provide new opportunities for catalytic processes and drug development. De novo protein design has emerged as a promising approach to create such enzymes, and the study of metalloproteins, particularly copper-binding peptides, has become a focus of this research. This thesis investigates the reactivity of a mutated copper-binding peptide, I5A-3SCC, with oxygen and its implications in the development of artificial enzymes.

The parent peptide, 3SCC, was mutated by replacing Isoleucine residues with smaller Alanine side chains, which was hypothesized to enhance the access of substrates into the active site. Various methods, including UV-Visible spectroscopy and kinetic experiments, were employed to analyze the peptide's properties and reactivity. The UV-Vis spectra of I5A-3SCC bound with copper displayed a broad d-d band absorption around 618 nm at pH 6.5, indicating copper binding in the active site. Furthermore, the reactivity of I5A-3SCC with oxygen was studied in the presence of ascorbic acid, revealing an increase in reactivity compared to the parent peptide, 3SCC. The formation of a copper-oxygen intermediate was observed, with a rate of 7.2 × 10-5 s-1, which was approximately 1.5 times higher than the parent peptide.

Additionally, the potential binding of phenol to the copper in ArCuPs was explored due to its relevance in the polyphenol oxidase reaction. A shift in the d-d band upon the addition of phenol suggested the occurrence of phenol binding, although further investigation is needed to confirm this interaction.

In conclusion, this thesis demonstrates the potential of de novo protein design for creating artificial enzymes by studying the reactivity of the I5A-3SCC copper mutant with oxygen. The findings suggest that the mutation of the parent peptide, 3SCC, results in increased reactivity in the presence of oxygen and ascorbic acid, as well as the formation of a copper-oxygen intermediate. Future research should focus on understanding the binding mechanism of phenol to the copper in ArCuPs and further exploring the potential applications of these peptides in enzyme design.

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