Mechanistic Study of the Urocanase Reaction Using Deuterated Substrates and 1H‐NMR Spectroscopy

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Abstract
Samples of (.alpha.-2H1, 5-2H1) and (.alpha.-2H1, .beta.-2H1) urocanic acid were prepared by a combination of chemical and enzymic methods. The enzymic conversion of unlabelled urocanate was followed by 1H-NMR spectroscopy at 500 MHz in deuterium oxide. It was found that [Pseudomonas putida] urocanase promotes the exchange of the 5-hydrogen atom of the substrate faster than it catalyses the overall reaction, that the product is an equilibrium mixture of racemic .beta.-(5-oxoimidazol-4-yl)propionate and .beta.-(5-hydroxyimidazol-4-yl)propionate and 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.degree. C. It was shown by UV and 1H-NMR spectroscopic measurements that .beta.-(5-hydroxyimidazol-4-yl)-propionate (.lambda.max .apprxeq. 234 nm) exists in protonated form at low pH (< 1), whereas at neutral pH (.apprxeq. 7.5) it exists in equilibrium with .beta.-(5-oxoimidazol-4-yl)propionate (.lambda.max .apprxeq. 269 nm). (.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. Kinetic deuterium isotope effects of between 2 and 3 were measured when the urocanase reaction was conducted in deuterium oxide at different p2H values. Implications of these findings for the mechanism of action of urocanase are discussed.