The Design of Allosterically Regulated Protein Catalysts
Date of Award
May 2016
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
Advisor(s)
Ivan V. Korendovych
Keywords
environmentally friendly chemistry, enzyme catalysis, enzyme promiscuity, green chemistry, minimalist approach, protein engineering
Subject Categories
Physical Sciences and Mathematics
Abstract
Producing novel catalytic function in proteins has intrinsic practical utility and provides a further understanding of enzymatic processes found in nature. Current state of the art computationally heavy methods have created enzymes with novel function, but are complex to utilize. However, minimalist approaches to create enzymes offer a more streamlined method that, when combined with several rounds of directed evolution can produce enzymes for various chemical reactions with comparable efficiency.
An enzyme was created through a minimalist approach to catalyze a benchmark reaction. This enzyme was created through introducing a single active amino acid residue and the enzyme’s catalytic efficiency was improved through several rounds of directed evolution 220 fold. Furthermore, the ability of the enzyme and its improved derivatives to catalyze a similar ring opening reaction has been characterized. This gives knowledge as to how enzymes designed by minimalist approaches can be sufficient starting points for similar chemical reactions as compared to enzymes designed by other approaches.
Additionally, minimalist approach was applied to create a retro-aldolase. Through strategic placement of a single active amino acid residue, a 15,000-fold increase in the second order rate constant was observed. This enzyme can be improved by completing rounds of directed evolution through screening of crude bacterial cell lysates. This demonstrates that the minimalist approach can be applied to chemical reactions which proceed by various mechanisms. All of the enzymes described above are allosterically regulated by calcium.
More recently, efforts are underway to engineer a manmade metalloprotein scaffold to catalyze oxidation reactions. A protein was constructed as a mimic of natural occurring enzymes and consists of a dimetal coordination sphere. The scaffold was redesigned to comprise of a single mononuclear metal center in order to catalyze oxidation reactions. This gives more coordination sites to accommodate a substrate and an oxidant. Finally, these enzymes designed by minimalist approaches act as catalytically amplified metal sensors, they can be utilized to sense various metals in biological processes and they provide means to more environmentally sustainable chemistry.
Access
Surface provides description only. Full text is available to ProQuest subscribers. Ask your Librarian for assistance.
Recommended Citation
Caselle, Elizabeth Anne, "The Design of Allosterically Regulated Protein Catalysts" (2016). Dissertations - ALL. 423.
https://surface.syr.edu/etd/423
