The photoregulation of urocanase purified from Pseudomonas putida was first demonstrated in this laboratory. The inactive enzyme was shown to be activated through a photodissociation of sulfite from the tightly-bound NAD+ coenzyme. We now propose to investigate the hypothesis that biochemically-generated electronically excited molecules can substitute for light in producing an activation. Preliminary results show that peroxidase (POD) catalyzed aerobic oxidation of indoleacetic acid (IAA), which produces idole-3-aldehyde in the excited triplet state, can produce an activationof the purified bacterial enzyme in the absence of light. We propose to use the IAA/POD/O2 system to study the mechanism of enzyme activation and determine how it compares to the photoactivation. Urocanase activity will be assessed spectrophotometrically and the potential involvement of reactive oxygen intermediates will be studied through the use of various traps and quenchers. Energy transfer to the urocanase-sulfite adduct can be established through quenching of chemiluminescence or eosine-sensitized fluorescence. Sulfite dissociation will be quantitated with a spectrophotometric method. Other preliminary results have shown that urocanase which is purified from beef liver can also be photoactivated. We will first determine whether the liver enzyme is regulated in the same manner as the bacterial enzyme, namely through sulfite addition and dissociation. Whether sulfite is the nucleophile responsible for inactivating urocanase in liver will be determined by purifying in vivo-inactivated enzyme and quantitating sulfite released upon photoactivation. We will then study activation of liver urocanase by triplet idole-3-aldehyde, which will serve as a model for putative excited state whose nature and origin are as yet unknown but which may exist in liver and other animal cells. The widespread occurence of bioluminescence in biology proves that excited molecules are relatively commonplace under physiological conditions. This process would represent a novel and previously unknown type of enzyme regulation. Knowledge of enzyme regulation is basic to an understanding of health and disease and therapeutic approaches to disease often can involve enzymes and their control. Regulation of urocanase activity may be involved in the regulation of histidine catabolism which produces histamine, a neurotransmitter and a hormone involved in allergic responses, as well as formiminoglutamate which provides one-carbon groups for purine synthesis. Urocanate itself has been identified in mammalian skin as the photoreceptor for photoimmune suppression.
