Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Characterization of Human Pyrroline-5-Carboxylate Reductase Enzymes Responsible for L-Proline Biosynthesis
Abstract
Pyrroline-5-carboxylate reductases (EC 1.5.1.2) are important housekeeping enzymes of L-proline biosynthesis, which generate L-proline and influence redox cycling of NAD(P)H/NAD(P)+ to support cellular growth in all domains of life. Structural evidence from X-ray crystal structures of HsPYCR1 (PDB codes 5UAT, 5UAU, and 5UAV) shows both NADPH bound in the N-terminal Rao-Rossmann fold motif and an important hydrogen bond or proton donor role for Thr238 with L-P5C. A Thr238Ala mutation results in a 10-fold loss in catalytic efficiency with varied L-P5C relative to the wild-type enzyme, thus indicating an important role for Thr238 during catalysis. For HsPYCR2, Henri-Michaelis-Menten kinetic analysis reveals a 164-fold loss in catalytic efficiency for the Arg119Cys mutant, with varied L-P5C and fixed NADH, relative to wild-type HsPYCR2. Profound effects on thermostability and secondary structure characteristics of the Arg251Cys mutant were determined by thermal shift assays and circular dichroism spectroscopy, respectively. Product(s) inhibition kinetics collectively indicate NADP+ and NAD+ are mixed noncompetitive inhibitors against NADPH and NADH, respectively, whereas L-proline is a competitive inhibitor against L-P5C. Taken together, these findings support a sequential-ordered binding enzyme mechanism of L-P5C binding first followed by NAD(P)H-binding. The ability of HsPYCR1 and 2 for reverse enzyme activity was observed with L-T4C as the reducing substrate. Structural evidence of a HsPYCR1─L-T4C binary complex, reverse direction saturation kinetics for both isozymes, and inhibition kinetics showing L-proline as a competitive inhibitor versus L-T4C, all indicate L-T4C shares the same active site as L-proline. Upon further evaluation of reverse direction reactions, we discovered association reactions between tris(alkyl)phosphine compounds and NAD+. Stopped-flow absorbance kinetics demonstrated rapid and reversible NAD+─tris(alkyl)phosphine nonenzymatic reactions with optimal absorbance at 334 nm for the reaction product. NMR spectroscopy identified a covalent adduct between the phosphorus of tris(2-carboxyethyl)phosphine or tris(3-hydroxypropyl)phosphine interacting at the C4 of the dihydronicotinamide ring of NAD+. Ultimately, this thesis dissertation provides strong structural and kinetic insights into human pyrroline-5-carboxylate reductase enzymes responsible for L-proline biosynthesis.
Subject Area
Biochemistry|Molecular biology|Molecular chemistry
Recommended Citation
Patel, Sagar Mahendrakumar, "Characterization of Human Pyrroline-5-Carboxylate Reductase Enzymes Responsible for L-Proline Biosynthesis" (2020). ETD collection for University of Nebraska-Lincoln. AAI28086040.
https://digitalcommons.unl.edu/dissertations/AAI28086040