The long term goal of this research is to apply techniques from protein chemistry, molecular biology, and x-ray crystallography to study thoroughly the mechanism, structure, and evolution of the enzymes in two meta-fission pathways-the catechol and homoprotocatechuate meta- fission pathways-the catechol and homoprotocatechuate meta-fission pathways. Such studies have implications for the understanding for the understanding of basic enzyme mechanisms, enol and dienol chemistry , and the evolution of enzyme specificity and catabolic pathways. In addition to addressing these intellectual questions, these studies may assist bio-remediation efforts. During this funding period, we will continue our studies on 4- oxalocrotonate tautomerase (4-OD), 4-oxalocrotonate decarboxylase (4- OD) and vinylpyruvate hydratase (VPH), three sequential enzymes found in the catechol metafission pathway. The proposed studies will address key mechanistic and evolutionary question raised by what appears to be a common in the catechol meta-fission pathway. The proposed studies will address key mechanistic and evolutionary pathways raised by what appears to be a common strategy used by the various meta-fission pathways to degrade catecholic compounds. Our major specific aims, listed in the order of priority, during this funding period will be to: (1) synthesize potential dicarboxylated inhibitors of 4- OT as ligands for crystal structures; (2) delineate the roles of the active site arginines in 4-OT using coded and non-coded amino acids; (3) delineate additional factors governing the reactivity of the catalytic base in 4-OT; (4) determine the 13C kinetic isotope effects on the non- enzymatic and enzymatic decarboxylation of 2-oxo-3-hexenedionate as a function of pH and medium; (5) investigate whether substrate channeling is involved in the 4-ODP/VPH complex; (6) synthesize and evaluate potential inhibitors of VPH; and (7) pursue crystallographic studies of 4- OD/VPH and OHED hydratase. These studies will address long-standing questions about these enzymes as well as new questions raised in the previous funding period. In the process, they will address major evolutionary questions concerning the enzymes of the catechol metafission pathway. The results from these studies serve as a basis for comparison to t he enzyme of other meta- fission pathways as well as the foundation for future experiments and ultimately the genetic manipulations of specificity and activity.