Thermal activation of photoactivatable urocanase from Pseudomonas putida.

The dark inactivation of urocanase from Pseudomonas putida is caused by the formation of a sulfite adduct of the tightly bound coenzyme, nicotinamide adenine dinucleotide. Photodissociation of this adduct by UV radiation restores the enzyme activity. Based on cold exhaustive dialysis the modification reaction appeared to be irreversible. However, we now report that sulfite modification of urocanase is reversible at higher temperatures. An Arrhenius plot of the thermal activation is linear (20-38 degrees C). The activation energy for the enzyme activation is 114 kJ mol-1. The substance that is photodissociated from inactive urocanase reacts with urocanase to reform the modified enzyme indicating that sulfite is not oxidized, or otherwise changed through these processes. Nucleophiles (sulfite, hydroxylamine, hydride, cyanide) are known to inhibit urocanase by forming adducts with nicotinamide adenine dinucleotide. Urocanase inactivated by hydride or cyanide is not reactivated thermally or photochemically. Urocanase inactivated by hydroxylamine and by glycylglycine can be reactivated by a thermal reaction. In conclusion, sulfite-modified urocanase, which is formed in cells, can be reactivated not only by sunlight but also at physiological temperatures.