MECHANISTIC STUDIES OF THE UROCANASE REACTION USING (1)H- AND (31)P-NMR SPECTROSCOPY AND THE SUBSTRATE ANALOGUE 2-METHYLUROCANATE

Article abstract of DOI:10.1016/S0040-4020(01)88662-2

The reaction of 2-methylurocanate with urocanase from Pseudomonas putida was monitored by (1)H-NMR spectroscopy at 500 MHz.The following conclusions were drawn: (i) 2-methylurocanate reacts 128 times more slowly with urocanase than does urocanate, (ii) no signals for the enol form of the produced 2-methylimidazolone propionate were detected, (iii) 2-methylimidazolone propionate is about 25 times more stable to hydrolysis than imidazolone propionate, (iv) the urocanase-catalysed exchange of the 5-proton of 2-methylurocanate with the solvent deuterium is 1.3 times faster than the overall reaction and (v) the non-enzymic exchange of the Me protons of 2-methylimidazolone propionate with solvent D takes place with a half life of 5.8 hr. By (1)H-NMR spectroscopy it was shown that the urocanase reaction is reversible.At 8^o and pD 6.3 1.6percent of the total imidazolone propionate was converted into urocanate. Apart from the pyrophosphate ester group of NAD(1+) no phosphorylated groups could be detected in urocanase by (31)P-NMR spectroscopy.