EVIDENCE FOR A CARBANION INTERMEDIATE IN THE ELIMINATION OF AMMONIA FROM L-HISTIDINE CATALYZED BY HISTIDINE AMMONIA-LYASE

Stable isotopically labeled histidines, i.e., L-[3,3,5'-2H3,3'-15N]histidine (His-3,3,5'-d3) and DL-[2,5'-2H2]histidine (His-2,5'-d2), were used as substrates to investigate the enzymatic reaction mechanism with histidine ammonia-lyase (EC 4.3.1.3) from Pseudomonas fluorescens in order to determine whether the elimination is concerted or involves a carbanion intermediate. The labeled L-histidine (His-3,3,5'-d3) (100 mM) was incubated with histidine ammonia-lyase (400 units) for 24 h at 25.0 °C (pH 9.0, 0.2 M Tris buffer) in the presence of 0.01 M MgCl2 and 0.1 M glutathione. The 400-MHz 1H NMR spectrum of the labeled urocanic acid product (UA-3,5'-d2) revealed the presence of a proton signal at δ 7.75 corresponding to the C-5' position of the imidazole ring, indicating the incorporation of hydrogen at C-5'. The extent of deuterium loss was calculated to be 44%, which was practically the same as that obtained from the mass spectral data (45.3%). When His-2,5'-d2 was incubated, the same extent of deuterium loss at C-5' was observed, being 42% based on the 1H NMR data. The evidence presented in this study supports a stepwise mechanism via a carbanion intermediate for the elimination of ammonia catalyzed by the enzyme. © 1990, American Chemical Society. All rights reserved.