Photoactivation of urocanase in Pseudomonas putida. Role of sulfite in enzyme modification.

Urocanase is inactivated in vivo and is reactivated by ultraviolet radiation. The chemistry of this photoregulation of urocanase was studied. Purified enzyme was inactivated by 25 pM sulfite (tl,z, 15 min). Dialyzed sulfite-treated inactive enzyme was photoactivatable. The activity can be cycled repeatedly by alternate ultraviolet light and sulfite treatments. Active enzyme binds 5 times more [35S]sulfite than inactive enzyme and 35S was released upon photoactivation of urocanase. Imidazolepropionate, a competitive inhibitor, protected the enzyme from sulfite inactivation, indicating that sulfite attacks the active site. NAD’ is the coenzyme; sulfite is known to react with free NAD+ to form an adduct which absorbs at 320 nm. The difference spectrum of inactive and active urocanase had a positive peak at 323 nm corresponding to a peak in the previously published action spectrum. The chromophores disclosed by the difference spectrum and by the action spectrum are probably the NAD-sulfite adduct. Inactive urocanase fluoresced at 430 nm when excited at 320 nm, but photoactivated enzyme did not. Excitation of inactive urocanase at 289 nm near the action spectrum maximum produced an emission peak at 335 run. The intensity of this emission peak was enhanced upon photoactivation of urocanase. This finding indicated that fluorescence of tryptophan residues was quenched by energy transfer from excited tryptophans to the NAD-sulfite adduct. A photochemical model was demonstrated consisting of dihydroxyacetone and acetylpyridine adenine dinucleotide. These results indicate that NAD+ bound in urocanase forms an adduct with sulfite which inactivates the enzyme; the adduct is photochemically dissociated by light absorbed at 323 nm or by energy transferred from a nearby chromophore, probably tryptophan; photodissociation of the NAD-sulfite adduct restores catalytic activity.