TYROSINE HYDROXYLASE--MECHANISTIC AND STRUCTURAL STUDIES

Texas A&M University System, College Station TX

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Abstract

DESCRIPTION The mechanistic and structural studies of the pterin-dependent metalloprotein tyrosine hydroxylase is the primary focus of this proposal. Tyrosine hydroxylase is the rate-limiting step in the biosynthesis of catecholamine neurotransmitters, and defects in neurotransmitter biosynthesis have been implicated in various neurological disorders. The crystal structure of the catalytic domain has been determined, and site directed mutagenesis of conserved amino acid residues located within the active site have been generated. The mutant proteins will be characterized to determine their effect on substrate binding, catalysis, and tetrahydropterin oxidation. A meta hydroxylated phenylalanine 300 was proposed to be involved in tetrahydropterin stabilization within the active site. The tryptic peptide containing the proposed hydroxylated phenylalanine 300 will be identified by mass spectrometric and peptide sequence analyses to determine if the purified native enzyme is hydroxylated. The role of the iron will be investigated through EPR analyses to determine if there is a specific coordination site located on the Fe(II) form of the enzyme for oxygen binding. The necessity for the iron in tetrahydropterin oxidation will also be tested with histidine mutant proteins previously shown to be iron-free. Although the structure for the catalytic domain is known, the structure of the full length enzyme has not been determined. Attempts will be made to obtain the structure of the phosphorylated and unphosphorylated full length enzyme with and without catecholamine bound.