TYROSINE HYDROXYLASE--MECHANISTIC AND STRUCTURAL STUDIES

描述蝶呤依赖性金属蛋白酪氨酸羟化酶的机制和结构研究是本提案的主要重点。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，神经递质生物合成的缺陷与各种神经系统疾病有关。催化结构域的晶体结构已被确定，并已产生位于活性位点内的保守氨基酸残基的定点诱变。突变蛋白将被表征以确定其对底物结合、催化和四氢蝶呤氧化的影响。提出Meta羟基化苯丙氨酸300参与活性位点内的四氢蝶呤稳定化。将通过质谱和肽序列分析鉴定含有拟定羟基化苯丙氨酸300的胰蛋白酶肽，以确定纯化的天然酶是否被羟基化。铁的作用将通过EPR分析进行研究，以确定是否有一个特定的配位位点位于Fe（II）形式的酶的氧结合。铁在四氢蝶呤氧化中的必要性也将用先前显示为不含铁的组氨酸突变蛋白进行测试。虽然催化结构域的结构是已知的，但全长酶的结构尚未确定。将尝试获得具有和不具有结合的儿茶酚胺的磷酸化和未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.