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

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 fi-elimination and gives pyruvate; this activity is elevated in colorectal cancer. This new NMR-based assay allows one to monitor both a-proton exchange chemistry and fi-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 similar to 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 fi-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 (fi-replacement: si-face protonation similar to 6:1 vs fi-elimination: si-face protonation similar to 1:3.6 for hSR), highlighting the value of this approach for fingerprinting the pyridoxal phosphate (PLP) enzyme mechanism.

Automated summary

The first dual-function assay for human serine racemase (hSR), which is essential for neuronal signaling and elevated in colorectal cancer, has been reported. This new NMR-based assay allows for monitoring of both a-proton exchange chemistry and fi-elimination, and it has been used to identify an inhibitor family that displays allosteric site binding behavior. Mechanistic information for hSR and a different partition ratio in the active site of the enzyme tryptophan synthase have also been revealed through this assay, demonstrating its value for studying enzyme mechanisms.