MECHANISTIC STUDIES OF THE UROCANASE REACTION USING (1)H- AND (31)P-NMR SPECTROSCOPY AND THE SUBSTRATE ANALOGUE 2-METHYLUROCANATE
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 8o 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.