Presence of tightly bound NAD+ in urocanase of Pseudomonas putida

Egan, R.M.; Phillips, A.T.

Journal of Biological Chemistry 252(16): 5701-5707

1977


ISSN/ISBN: 0021-9258
PMID: 18470
Accession: 068518108

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Abstract
The reduction of urocanase [EC 4.2.1.49] with NaB3H4 plus NaB2H4 produced an inactive enzyme upon incorporation of 1 g atom of H/mol of enzyme. From acid hydrolysates of this reduced enzyme was isolated labeled nicotinic acid which was identifed by chromatography, electrophoresis and mass spectral analysis. The presence of a nicotinic acid-containing component was confirmed by incorporation of [14C]nicotinic acid into urocanase during growth of a nicotinic acid auxotroph of P. putida. Treatment of this purified [14C]urocanase with perchloric acid released a nicotinic acid-containing cofactor which was subsequently determined to be NAD+ by electrophoresis and enzymatic analysis. Incorporation of 3H upon NaB3H4 reduction of urocanase, microbiological analysis for nicotinic acid and the specific radioactivity of [14C]urocanase all supported a stoichiometry of 1 mol of NAD+ bound/mol of native enzyme. The necessity of using denaturing conditions to remove enzyme-bound NAD+ indicated that the NAD+ is very tightly bound. The principal criteria employed earlier for the identification of .alpha.-ketobutyrate as a prosthetic group in urocanase, namely similar chromatographic and electrophoretic properties for .alpha.-hydroxybutyrate and 3H material isolated from urocanase after NaB3H4 reduction and acid hydrolysis, were unable to distinguish between these compounds and nicotinic acid, thereby accounting for the incorrect conclusion that .alpha.-ketobutyrate was an essential cofactor. Apparently, NAD+ is a functional coenzyme in urocanase and probably catalysis proceeds through an internal oxidation-reduction mechanism.