Mechanism of Action of Urocanase: Ruling Out a 1,5 Sigmatropic H-Shift in the Intermediate NAD+ Urocanate Adduct

The tightly bound NAD+ in urocanase forms a covalent adduct with the inhibitor imidazole propionate and most likely also with the substrate urocanate. Subsequent tautomerizations and cleavage of the adduct lead eventually to the product, 5-hydroxyimidazole propionate. The possibility that the H4 atom of the nicotinamide moiety undergoes a 1,5 sigmatropic H-shift in one of the intermediates has been investigated by deuterium labeling. No incorporation of solvent deuterium into position 4 of the nicotinamide moiety could be detected, which rules out a 1,5-sigmatropic shift of the H4 atom.