Targeting a Unique Cysteine Residue to Achieve Isoform-Selective Inhibition of the Proline Biosynthetic Enzyme Pyrroline-5-Carboxylate Reductase 2

value of 22 nM, which is 10-fold more sensitive than with PYCR1. Results from protection assays with dithiothreitol, site-directed mutagenesis, and mass spectrometry implicate Cys232 in PYCR2 as the target of ebselen. A new crystal structure of PYCR2 shows that Cys232 is in the P5C-binding loop, whereas PYCR1 contains a serine at this position. Our study provides new insight into the structural and functional roles of unique cysteine residues in PYCR2. Further, our results demonstrate proof-of-concept for targeting a noncatalytic cysteine as a new approach for selectively inhibiting PYCR2 over PYCR1.