REVERSIBLE-REACTION VIA A CARBANION INTERMEDIATE IN THE ELIMINATION OF AMMONIA FROM L-HISTIDINE CATALYZED BY HISTIDINE AMMONIA-LYASE

L-[5'-H2(H-2)]Histidine was used as a substrate to investigate the enzymatic reaction mechanism with histidine ammonia-lyase from Pseudomonas fluorescens. The study was performed to determine the exchange rate of deuterium at C-5' of the imidazole ring with solvent hydrogen relative to the net urocanic acid production. The extent of hydrogen exchange at C-5' of histidine or urocanic acid was measured by gas chromatography-mass spectrometry-selected ion monitoring, monitoring the molecular ion intensities of the respective gas chromatographic derivatives, at m/z 380 and 379 for histidine and at m/z 267 and 266 for urocanic acid. The observed hydrogen exchange at C-5' suggested a reversible mechanism via a carbanion intermediate in the reaction with histidine ammonia-lyase.