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Kinetic analysis of the multifunctional proline utilization a (PutA) flavoprotein from Escherichia coli

Michael A Moxley, University of Nebraska - Lincoln


The primary objective of this dissertation has been to systematically describe the enzyme kinetic attributes of the multifunctional proline utilization A (PutA) flavoprotein from Escherichia coli (EcPutA). The work described here, however, not only has implications with EcPutA directly but can be used to understand proline metabolic enzymes in general in nearly any organism. The introduction outlines the analysis of kinetic data which stretches back to the beginning of the twentieth century but yet is still being modified even today to extract as much information as possible from enzyme kinetic data. The introduction has included analysis using both steady-state and rapid reaction methods along with a strong introduction of the current knowledge of EcPutA and its homologs. Subsequent chapters describe the delineation of the steady-state mechanism (chapter 2), identification of microscopic rate constants (chapter 3), and a critical residue involved in multiple EcPutA functions (chapter 4). Chapter 5 addresses a number of residues hypothesized to be involved in membrane and ubiquinone binding however further studies are needed. Chapter 6 includes a brief study of human pyrroline-5-carboxlyate dehydrogenase that is useful for understanding the bienzymatic properties of EcPutA. Finally in chapter 7, preliminary results for EcPutA channeling experiments are given and show a first look at a bienzymatic kinetic mechanism with potential to describe the overall proline and P5C dehydrogenase activity of EcPutA. From a global point of view, data collected for this dissertation could be used to help understand proline metabolism and beyond from a quantitative perspective. This perspective is required for a detailed time resolved understanding of biological processes.

Subject Area


Recommended Citation

Moxley, Michael A, "Kinetic analysis of the multifunctional proline utilization a (PutA) flavoprotein from Escherichia coli" (2012). ETD collection for University of Nebraska - Lincoln. AAI3504203.