9603874 Libby Part 1-Technical The prevailing model for heme enzyme catalysis ascribes variations in mechanism entirely to characteristics of the protein and ignores effects of variation in substrate reactivity. The specific research plan proposed investigates the effects of substrate structure on the pathway by which it is consumed by chloroperoxidase, a versatile heme enzyme. First, reactions will be characterized as to products and their distributions. Second, the kinetics of each process will be probed to determine (1.) effects of one substrate type on another and (2.) inhibitory effects of trapping agents for reactive species (e.g. molecular halogens or free radicals). Finally, the redox potentials of substrates will be determined. The focus of the analyses will be on independent determination of substrate steric and electronic effects on the extent of involvement of reactive non-enzymic intermediates in chloroperoxidase reactions. A number of trapping agents for enzyme generated intermediates will be identified and characterized. These agents should be generally useful for detecting significant involvement of enzyme generated oxidative species. Thus, our results should help elucidate mechanisms of chloroperoxidase catalyzed reactions, and also provide a series of new inhibitors for probing reaction mechanisms of many other oxidative enzymes. Part 2-Non Technical Hemoproteins have a diverse variety of functions in living organisms, including carrying of molecular oxygen, oxidation. Chloroperoxidase (CPO) is a heme containing enzyme that has chlorination, peroxidation and catalase activities. Strategies are described to elucidate the factors responsible for partitioning among the various reaction mechanisms in the reactions catalyzed by CPO. CPO has been studied for over twenty years, yet little is known about how each type of activity relates to the others. This research will play a significant role in our understanding of CPO and oxidative catalysis of heme enzymes in general.
