The catalytic properties of iodotyrosine deiodinase will be examined to identify the source of its unique reactivity. This enzyme is one of only two types of mammalian enzymes known to effect reductive iodide elimination and thus it provides a superb model for investigating the chemical basis of metabolic diversity within organisms. The rare but physiological amino acids, mono- and diiodotyrosine, are specifically activated and transformed in the thyroid by the deiodinase to yield iodide and tyrosine. This process serves as the critical salvage pathway for iodide and thus is essential for human health. The dehalogenation of other potential substrates has not yet been investigated but may play a role in the endogenous metabolism of halogenated pollutants. The deiodinase is dependent on an enzyme-bound flavin of unprecedented activity. Consequently, the chemical elaboration of this reaction will expand the known repertoire of flavin-based transformations. The investigators description of catalysis will begin with the analysis of the aromatic substitution reaction essential for deiodination. Activation of this process will be measured in part by the interaction of the diodinase with a range of substituted tyrosine derivatives. Additional enzymological study include the use of: (1) reversible enzyme inhibition to define both substrate recognition patterns and catalytic transition-state (reactive intermediate) properties and (2) mechanism-based inactivation to characterize the activation of tyrosine derivatives. To focus on the pharmacologically important protein chemistry of this dehalogenation system, new methods for purifying and reconstituting the deiodinase will also be assessed. Isolation of homogeneous enzyme in large quantities, while not required for most studies proposed, will be necessary for future investigations. Similarly, methods for continuous assay of enzyme turnover will be examined in order to facilitate later kinetic analyses. The novel properties revealed for this unusual catalyst should contribute to the underlying goal of finding new targets for therapeutic control of thyroid disease.
