Mechanistic study of the urocanase reaction using deuterated substrates and 1H-NMR spectroscopy

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.