Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. This pivotal enzyme is under strict regulatory control and responds to a variety of cellular stimuli by increasing its activity. In the short-term, TH is phosphorylated in vivo by at least three separate protein kinase systems (cAMP-dependent protein kinase, Ca++/phospholipid- dependent protein kinase and CA++/calmodulin-dependent protein kinase). TH can be phosphorylated in vitro by two additional kinases (cGMP-dependent protein kinase and TH-associated protein kinase from pheochromocytoma cells). Although the individual contributions of the separate kinases are unclear, the overall consequence of phosphorylation is an increase in enzyme activity. Five serine residues in rat pheochromocytoma TH have been identified as sites of phosphorylation. We propose to utilize a full-length cDNA clone (isolated in this laboratory) to test the functional significance of the phosphorylation of these serine residues. This cDNA clone has been expressed in bacteria as two different forms of beta-galactosidase fusion proteins. Both of these fusion enzymes exhibit high levels of activity. Attempts will be made to express TH as a native enzyme within bacteria. These recombinant enzymes will be purified and characterized as to their physical state, enzyme kinetics parameters and phosphorylation by protein kinases. Site specific mutagenesis will be employed to convert serine residues to a number of different amino acids. The altered enzymes will then be expressed and tested from their ability to be phosphorylated and activated by protein kinases. The role of each serine in TH activity and its post- translational regulation will be established. Deletion mutants will be constructed from each end of the TH cDNA. The prevailing hypothesis that TH is composed of an amino terminal regulatory domain and carboxyl terminal catalytic domain will be tested. This will also delineate the boundaries of the catalytic core of the enzyme. Both deletion and site specific point mutants will be constructed to test the hypothesis that leucine zippers are involved in the assemble of TH monomers into its native homotetramer form. We have identified two strong candidates for leucine zippers (with a third weaker candidate) in the carboxyl terminus of TH. These will be examined by interrupting the leucine repeat which is characteristic of the leucine zipper, expressing the enzyme and determining if it associates into multimers.
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