<p> Site-directed mutagenesis provides a powerful tool in the study of enzyme function. Residues suggested to be important for catalysis can be readily mutated and the energetic effects measured. However, the limited repertoire of naturally occurring amino acids constrains the substitutions that can be made. To obtain a deeper understanding of how enzymes work requires using unnatural amino acids to systematically perturb enzymatic residues. For example, hydrogen bonds in an ‘oxyanion hole’ are a common feature of enzyme active sites and often suggested to be important for catalysis. However, water can form hydrogen bonds, so for enzyme-mediated hydrogen bonds to be catalytic the energetics of these hydrogen bonds must be different than those made with water. A previous study in the enzyme ketosteroid isomerase (KSI) used a series of fluorotyrosine analogs to perturb the p<i>K<sub>a</sub></i> of the tyrosine hydrogen bond donor and results suggested a modest catalytic contribution of oxyanion hole hydrogen bonds. However, challenges in synthesis limited the set of fluorotyrosine analogs used. To overcome these challenges and extend the series of fluorotyrosines used in enzymatic studies, we developed an approach to selectively incorporate fluorotyrosines in peptides using silyl-based protecting groups. The fluorotyrosine must be protected on the amino and phenol groups and then incorporated in a peptide using solid phase synthesis. More so, the protection chemistry must be friendly in such a way that it does not have drastic side effects on any other part of the system. We tested the stability of silyl groups including TBDMS-Cl, TIPS-Cl, and TBDPS-Cl for their use in peptide synthesis. With the doubly protected fluorotyrosines, solid phase peptide synthesis will occur in order to place the new tyrosine into the protein of interest where these analogs will be used to investigate the energetics of enzymatic hydrogen bonds.</p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:1603748 |
| Date | 26 November 2015 |
| Creators | Colla, Alexander J. |
| Publisher | California State University, Long Beach |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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