Biochemistry, Department of
Investigation of Proline Utilization A: Kinetic Analysis of Substrate Channel-blocking Mutants and Creation of a Trifunctional Chimera Enzyme
Date of this Version
Arentson, BW. (2013). Investigation of Proline Utilization A: Kinetic Analysis of Substrate Channel-blocking Mutants and Creation of a Trifunctional Chimera Enzyme. (Doctoral Dissertation).
Proline metabolism is known to be involved in many cellular processes such as cell signaling, cellular redox balance, and cell survival. One of the enzymes involved in proline catabolism, proline utilization A, plays a role in oxidizing proline to glutamate in a two-step oxidation pathway involving enzymes proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH).
Intermediate P5C/GSA has been shown to use an intramolecular channel to move from the PRODH active site to the P5CDH active site in a phenomenon called substrate channeling. In this work, one of the main objectives was to learn more about the channel usage. Chapter 2 demonstrates that making mutations along the channel can impede passage of intermediate, and helps describe how P5C accesses the P5CDH domain.
A second objective was to gain understanding of the structure-function relationship of the DNA-binding domain of trifunctional PutAs. Chapter 3 discusses how a chimera enzyme was created by attaching the DNA-binding domain of a trifunctional PutA to a bifunctional PutA in order to make an artificial trifunctional PutA. These results help to better understand how the DNA-binding domain orientation is important and provide clues that more residues from the DNA-binding domain may be necessary for DNA-binding in vivo.
Chapter 4 explores a new ubiquinone analog, and provides kinetic evidence suggesting it is a substrate for the PRODH domain. Additionally Geobacter sulfurreducens was kinetically characterized and was shown to substrate channel.
Collectively, this dissertation aims to provide a further understanding of usage of the substrate channel in proline oxidation and insight into the structure-function relationship of trifunctional PutAs and functional switching.
Advisor: Donald F. Becker
A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biochemistry, Under the Supervision of Professor Donald F. Becker. Lincoln, Nebraska: November 2013
Copyright (c) 2013 Benjamin W. Arentson