Mechanism of action of urocanase. Specific 13C-labelling of the prosthetic NAD+ and revision of the structure of its adduct with imidazolylpropionate.
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.
Klepp
J
J
Lehrstuhl für Biochemie, Universität Karlsruhe, Federal Republic of Germany.
Fallert-Müller
A
A
Grimm
K
K
Hull
W E
WE
Rétey
J
J
D001665:Binding Sites / Q000187:drug effects; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology*; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D009525:Niacin / Q000494:pharmacology; D011422:Propionates / Q000737:chemistry / Q000494:pharmacology*; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D014559:Urocanate Hydratase / Q000037:antagonists & inhibitors / Q000737:chemistry*
D007093:Imidazoles; D011422:Propionates; D009243:NAD; C060786:3-(2-imidazo(4,5-b)pyridine)propionic acid; D009525:Niacin; D014559:Urocanate Hydratase