The stoichiometry of the tightly bound NAD+ in urocanase. Separation and characterization of fully active and inhibited forms of the enzyme.
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.
Klepp
J
J
Lehrstuhl für Biochemie im Institut für Organische Chemie der Universität Karlsruhe, FRG.
Rétey
J
J
D001665:Binding Sites / Q000528:radiation effects; D001894:Borohydrides / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006836:Hydro-Lyases / Q000302:isolation & purification*; D009243:NAD / Q000032:analysis*; D009525:Niacin / Q000032:analysis; D010777:Photochemistry; D011549:Pseudomonas / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D014559:Urocanate Hydratase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000528:radiation effects
D001894:Borohydrides; D009243:NAD; D009525:Niacin; C025364:sodium borohydride; D006836:Hydro-Lyases; D014559:Urocanate Hydratase