ABSTRACT
1. [4-13C]Nicotinate was synthesised and used to support the growth of a nicotinate auxotrophic mutant of Pseudomonas putida. 13C-NMR spectroscopy of the isolated urocanase confirmed the efficient incorporation of 13C into C4 of the nicotinamide ring of the tightly bound NAD+ cofactor. 2. beta-[( 2'-13C]Imidazol-4-yl)propionate was synthesised according to known procedures and used for inhibition of the 13C-labelled urocanase. An increase in the absorbance at 330 nm indicated adduct formation between enzyme-bound NAD+ and inhibitor. The adduct was stabilised by oxidation with phenazine methosulfate and isolated using a slight modification of the procedure of Matherly et al. [Matherly, L. H., DeBrosse, C. W. & Phillips, A. T. (1982) Biochemistry 21, 2789-2794]. 3. The 13C-NMR spectrum of the doubly labelled adduct, [4-13C]NAD-[2'-13C]imidazolylpropionate, showed no one-bond 13C-13C coupling between labelled sites. The 1H-NMR spectrum of this adduct in 2H2O showed only one imidazole signal, which appeared as a doublet (1JC-H = 212 Hz), confirming the presence of a proton at the labelled C2'. The lack of a C5' signal and further NMR data provide evidence for a C-C bond between C4 of the nicotinamide and C5' of the imidazole ring. 4. The revised structure for the enzymatically formed addition complex suggests a novel mechanism for the urocanase reaction which is not only chemically plausible but also explains the previously observed urocanase-catalysed exchange of the C5 proton of urocanate and of beta-(imidazol-4-yl)propionate.
