The stoichiometry of the tightly bound NAD+in urocanase

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Abstract
Urocanase, purified by classical methods [Keul, V., Kaeppeli, F., Ghosh, C., Krebs, T., Robinson, J. A. and Rétey, J. (1979)J. Biol. Chem. 254, 843–851] fromPseudomonas putidawas submitted to high‐performance liquid chromatography on a TSK‐DEAE column. The enzyme was eluted in three resolved peaks (A, B and C) exhibiting specific activities of 3.4 U/mg, 1.85 U/mg and 0.4 U/mg, respectively. The difference spectra of peaks B and A as well as of C and A showed maxima at 330 nm. Irradiation of peaks B and C at 320 nm resulted in an increase of urocanase activity by 45% and 400%, respectively. Peak A could not be photoactivated. Rechromatography of the photoactivated peaks B and C on the TSK‐DEAE column confirmed their partial transformation into peak A. Spectroscopic methods for quantitative protein determination were adapted to urocanase. The stoichiometry of bound NAD+/urocanase (form A) was determined to be 1.75 by enzymic analysis of the free NAD+released upon acid denaturation of the holoenzyme. A similar stoichiometry (1.8–1.9) was found for all three forms (A, B and C) by biosynthetic incorporation of [7‐14C]nicotinate into urocanase using a nicotinate auxotrophic mutant ofP. putida. Form A of urocanase showed, after treatment with NaBH4up to 50% inhibition, an elution pattern (TSK‐DEAE column) similar to a mixture of forms A, B and C in the approximate ratio of 1:2:1. None of these forms could be photoactivated. We conclude that form A of the urocanase dimer contains two intact NAD+molecules. In form B one of the two subunits contains an NAD+‐nucleophile adduct which is present in both subunits of form C. Full urocanase activity requires intact NAD+in both subunits. Intact NAD+can be regenerated from the adduct but not from the reduced form by photolysis. The two subunits of urocanase are independent both in their catalytic activity and in modification reactions.