4-Hydroxyphenylpyruvate Dioxygenase and Tyrosinemia

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DESCRIPTION (provided by applicant): Iron-dependent non-heme oxygenases are the specific focus of this research. These enzymes catalyze vital metabolic and catabolic reactions throughout mammalian metabolism. The initial intent is to develop a fundamental understanding of the chemistry of these enzymes by the identification of the transient oxygen intermediates that are generated during catalysis. The long-term objective is to find evidence for the geometry of transition states for specific catalytic steps such that stable transition state mimics can be developed as inhibitory therapeutic agents to control the flux through key metabolic pathways. Initially, the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) has been selected for investigation. This enzyme exemplifies many of the catalytic functions of other FeII-dependent non-heme dioxygenases such as aromatic oxygenation, oxidative decarboxylation and substituent migration. Furthermore, it is one of the few alpha-keto acid-dependent oxygenases for which the crystal structure is known. Moreover, it has become a paradigm example of the ability to alleviate metabolic disorders through selective enzymatic inhibition. Type 1 Tyrosinemia, is caused by a deficiency of active fumarylacetoacetase, an enzyme that catalyzes the final step in the pathway for the catabolism of tyrosine. In the absence of treatment this disease is often fatal in the first year of life and always prior the completion of the first two decades. It is known, however, that the inhibition of HPPD which catalyzes the second step of this pathway is, in most cases, an effective treatment for type 1 Tyrosinemia. Part of the intent of this research is that the mechanism of this inhibition be understood in the context of catalytic events of the enzyme. The experimental approach will be to combine steady state and pre-steady state analyses with the selective use of substrate analogs, isotopic labels and mutagenesis and crystallography to test mechanistic hypotheses on the basis of rate constant modulation and structure.