Information-Rich, Dual-Function 13 C/ 2 H-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 2023-02, Vol.145 (5), p.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: Article
• Language: English
• Subjects: Humans; Protons; Pyridoxal Phosphate; Racemases and Epimerases; Serine - chemistry
• 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 -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 C-labeling and C/ H 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 -face much faster than does S84 on the -face. Moreover, the C NMR peak pattern seen is suggestive of internal return, pointing to K56 as the likely enamine-protonating residue for β-elimination. The C/ H-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: -face protonation ∼6:1 vs β-elimination: -face protonation ∼1:3.6 for hSR), highlighting the value of this approach for fingerprinting the pyridoxal phosphate (PLP) enzyme mechanism.