Information-Rich, Dual-Function 13C/2H‑Isotopic Crosstalk NMR Assay for Human Serine Racemase (hSR) Provides a PLP-Enzyme “Partitioning Fingerprint” and Reveals Disparate Chemotypes for hSR Inhibition

• Published in: Journal of the American Chemical Society Vol. 145; no. 5; pp. 3158 - 3174
• Main Authors: Ramos de Dios, Stephany M., Hass, Jared L., Graham, Danielle L., Kumar, Nivesh, Antony, Aina E., Morton, Martha D., Berkowitz, David B.
• Format: Journal Article
• Language: English
• Published: American Chemical Society 08.02.2023
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.2c12774

The first dual-function assay for human serine racemase (hSR), the only bona fide racemase in human biology, is reported. The hSR racemization function is essential for neuronal signaling, as the product, d-serine (d-Ser), is a potent N-methyl d-aspartate (NMDA) coagonist, important for learning and memory, with dysfunctional d-Ser-signaling being observed in some neuronal disorders. The second hSR function is β-elimination and gives pyruvate; this activity is elevated in colorectal cancer. This new NMR-based assay allows one to monitor both α-proton-exchange chemistry and β-elimination using only the native l-Ser substrate and hSR and is the most sensitive such assay. The assay judiciously employs segregated dual 13C-labeling and 13C/2H crosstalk, exploiting both the splitting and shielding effects of deuterium. The assay is deployed to screen a 1020-compound library and identifies an indolo-chroman-2,4-dione inhibitor family that displays allosteric site binding behavior (noncompetitive inhibition vs l-Ser substrate; competitive inhibition vs adenosine 5′-triphosphate (ATP)). This assay also reveals important mechanistic information for hSR; namely, that H/D exchange is ∼13-fold faster than racemization, implying that K56 protonates the carbanionic intermediate on the si-face much faster than does S84 on the re-face. Moreover, the 13C NMR peak pattern seen is suggestive of internal return, pointing to K56 as the likely enamine-protonating residue for β-elimination. The 13C/2H-isotopic crosstalk assay has also been applied to the enzyme tryptophan synthase and reveals a dramatically different partition ratio in this active site (β-replacement: si-face protonation ∼6:1 vs β-elimination: si-face protonation ∼1:3.6 for hSR), highlighting the value of this approach for fingerprinting the pyridoxal phosphate (PLP) enzyme mechanism.