TYROSINE HYDROXYLASE--MECHANISTIC AND STRUCTURAL STUDIES

说明依赖于蝶呤的金属蛋白酪氨酸羟基酶的机制和结构研究是这项建议的主要重点。酪氨酸羟化酶是儿茶酚胺类神经递质生物合成的限速步骤，神经递质生物合成缺陷与多种神经系统疾病有关。已经确定了催化区的晶体结构，并对位于活性中心的保守氨基酸残基进行了定点突变。突变的蛋白质将被鉴定以确定它们对底物结合、催化和四氢蝶呤氧化的影响。一个间羟基苯丙氨酸300被认为参与了四氢蝶呤在活性部位的稳定作用。含有所建议的羟基苯丙氨酸300的胰解肽将通过质谱学和多肽序列分析进行鉴定，以确定纯化的天然酶是否羟化。铁的作用将通过EPR分析来研究，以确定是否在酶的Fe(II)形式上存在用于氧结合的特定配位。铁在四氢蝶呤氧化中的必要性也将用以前被证明是无铁的组氨酸突变蛋白来测试。虽然催化区的结构已知，但全长酶的结构尚未确定。将尝试获得结合和不结合儿茶酚胺的磷酸化和非磷酸化全长酶的结构。

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.