Kinetics of human pyrroline-5-carboxylate reductase in l-thioproline metabolism

l-Thioproline (l-thiazolidine-4-carboxylate, l-T4C) is a cyclic sulfur-containing analog of l-proline found in multiple kingdoms of life. The oxidation of l-T4C leads to l-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of l-T4C to l-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and l-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with l-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant l-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M−1 s−1) than PYCR1 (13.7 M−1 s−1). Interestingly, no activity was observed with either l-Pro or the analog dl-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize l-T4C as a substrate. Inhibition kinetics show that l-Pro is a competitive inhibitor of PYCR1 KICapp=15.7mM\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left({K_{IC}^{app}}=15.7 \,{\rm mM} \right)$$\end{document} with respect to l-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that l-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of l-T4C.