[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.
β‐([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].
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.
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 β‐(imidazol‐4‐yl)propionate.
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