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1. Samples of (alpha-2H1, 5-2H1) and (alpha-2H1, beta-2H1) urocanic acid were prepared by a combination of chemical and enzymic methods. 2. The enzymic conversion of unlabelled urocanate was followed by 1H-NMR spectroscopy at 500 MHz in deuterium oxide. It was found (a) that urocanase promotes the exchange of the 5-hydrogen atom of the substrate faster than it catalyses the overall reaction, (b) that the product is an equilibrium mixture of racemic beta-(5-oxoimidazol-4-yl)propionate and beta-(5-hydroxyimidazol-4-yl)propionate and (c) that beta-(5-oxoimidazol-4-yl)-propionate is spontaneously hydrolysed under physiological conditions to N-formylisoglutamine. The rate of this hydrolysis is considerably diminished at +8 degrees C. 3. It was shown by ultraviolet and 1H-NMR spectroscopic measurements that beta-(5-hydroxyimidazol-4-yl)-propionate (gamma max approximately equal to 234 nm) exists in protonated from at low pH (less than 1) whereas pH (approximately equal to 7.5) it exists in equilibrium with beta-(5-oxoimidazol-4-yl)propionate (gamma max approximately equal to 269 nm). 4. (alpha-2H1, beta-2H1)Urocanate was reacted with urocanase in deuterium oxide. 1H-NMR spectroscopy at 500 MHz showed a slight incorporation of protium into the side-chain of the product. The incorporated protium corresponded roughly to the protium contamination of the solvent and was equally distributed between the alpha and beta positions. No transfer of the 5-hydrogen atom to the side-chain was detected. 5. Kinetic deuterium isotope effects of between 2 and 3 were measured when the urocanase reaction was conducted in deuterium oxide at different p2H values. 6. Implications of these findings for the mechanism of action of urocanase are discussed.

Incubation of urocanase with 2-methylurocanate leads, after an initial normal reaction, to a time dependent inactivation of the enzyme. It is suggested that a tautomeric form of the product, 2-methyl-imidazolone propionate, is the actual inhibitor. On the basis of these and of published experimental data a novel mechanism is proposed for the urocanase reaction. The crucial and initial step is the electrophilic addition of enzyme-bound NAD to the 2-position of the imidazole nucleus of urocanate.