Structural and kinetic studies of proline metabolic enzyme inhibitors and inactivators
Abstract
Structural and kinetic studies of the proline metabolic enzymes proline dehydrogenase (PRODH), [delta]1-pyrroline-5-carboxylate reductase (PYCR), and aldehyde dehydrogenase 4A1 (ALDH4A1) are presented in this thesis. Identifying inhibitors and inactivators of these enzymes can be purposed for chemical probes in studies elucidating the role of proline metabolism in cancer and other diseases. Chapter 1 introduces the enzymes of proline metabolism and provides a summary of literature on the involvement of PYCR isoforms in cancer. Additionally, a compilation of RNA transcript data was used to explore differential gene expression of PYCRs across 28 cancer types. Chapter 2 details a structural and enzyme kinetic investigation of compounds that noncovalently bind within the PRODH active site. These data provide insight into structure-affinity relationships of the best-known PRODH inhibitor, L-tetrahydro-2-furoic acid. Chapter 3 focuses on the inhibition of the proline/hydroxyproline catabolic enzyme ALDH4A1 by the stereoisomers of proline and 4-hydroxyproline. We found that hydroxyproline catabolism is subject to substrate inhibition by trans-4-hydroxy-L-proline, analogous to the known inhibition of proline catabolism by L-proline. Finally, following on our recent discovery of photoinduced covalent inactivation of PRODH by 1,3-dithiolane carboxylate, Chapter 4 preliminarily explores the scope of this reaction using seven acyclic carboxylic acids that contain an S atom bonded to the a-carbon, a feature we hypothesize is important for inactivation. Crystallographic evidence for three of these compounds covalently and irreversibly inactivating the flavin of PRODH was obtained. Also, a structure of one compound noncovalently bound in the active site was determined, which represents the initial step of the inactivation mechanism. Four compounds showed signs of reversibility, which is a new feature of this class of inactivator and remains to be explored.
Degree
Ph. D.