A covalent nicotinamide adenine dinucleotide intermediate in the urocanase reaction

• Published in: Biochemistry (Easton) Vol. 21; no. 11; pp. 2789 - 2795
• Main Authors: Matherly, L H, DeBrosse, C W, Phillips, A T
• Format: Journal Article
• Language: English
• Published: United States 01-01-1982
• Subjects: Chemical Phenomena; Chemistry; fluorescence; Hydro-Lyases - metabolism; Imidazoles; Kinetics; Magnetic Resonance Spectroscopy; N.M.R; NAD - metabolism; Propionates; Pseudomonas - enzymology; Pseudomonas putida; Spectrometry, Fluorescence; urocanate dehydratase; Urocanate Hydratase - metabolism
• ISSN: 0006-2960
• DOI: 10.1021/bi00540a033

When imidazolepropionate, a competitive inhibitor of the nicotinamide adenine dinucleotide (NAD) dependent urocanase from Pseudomonas putida , binds to the enzyme, a chromophore is produced that exhibits an absorption maximum near 330 nm. In order to establish the identity of this chromophore, and particularly to determine whether this material might be enzyme-bound reduced NAD (NADH) formed by a hydride ion transfer from imidazolepropionate, fluorescence studies of the imidazolepropionate-enzyme complex were undertaken, along with isolation of the pyridine nucleotide coenzyme from the enzyme. Detergent treatment of the complex released a super(14)C-labeled coenzyme that was then oxidized with phenazine methosulfate and purified by gel filtration and ion-exchange chromatography, whereupon it was established to be an addition product of NAD super(+) and imidazolepropionate. Proton nuclear magnetic resonance spectroscopy of the oxidized pyridine nucleotide derivative revealed an attachment of the tau nitrogen of the imidazole ring of the analogue to the 4 position of the nicotinamide portion of NAD super(+).