The stoichiometry of the tightly bound NAD+ in urocanase. Separation and characterization of fully active and inhibited forms of the enzyme

Abstract

1. 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] from Pseudomonas putida was 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. 2. The difference spectra of peaks B and A as well as of C and A showed maxima at 330 nm. 3. 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. 4. 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 of P. putida. 5. Form A of urocanase showed, after treatment with NaBH4 up 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. 6. 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.