Mechanisms of Neurotransmitter Biosynthesis

说明(申请人提供)：苯丙氨酸羟基酶、酪氨酸羟基酶和色氨酸羟基酶组成非血红素铁依赖的芳香氨基酸羟基酶家族。每种酶都催化底物芳香族侧链的羟化反应，这一反应对中枢神经系统的正常运作至关重要：苯丙氨酸代谢、儿茶酚胺生物合成和5-羟色胺生物合成。除了它们的生理重要性之外，这些酶还是独一无二的，因为它们是唯一的非血红素铁酶，其生理反应是未活化的芳香族系统的羟基化。本提案涉及关于这些酶的催化和调节机制的几个悬而未决的问题。将使用快速反应方法和光谱相结合的方法来研究共同的催化机理，目的是表征催化中的个别铁物种。通过测定底物对肽键与溶剂交换动力学的影响，研究蛋白质动力学在酪氨酸和苯丙氨酸羟基酶催化中的作用。苯丙氨酸和酪氨酸羟基酶的调节对它们的正常功能至关重要。肽键交换动力学将被用来评价苯丙氨酸羟基酶变构调节的文献模型，而荧光方法将被用来评价通过磷酸化调节酪氨酸羟基酶的结构模型。这些酶中的活性部位铁原子被2-组氨酸-1-羧酸三联体结合。苯丙氨酸羟基酶中的这两个组氨酸将被类似物取代，并结合稳态动力学、产物分配和动力学同位素效应来确定对个别动力学步骤的影响。芳香氨基酸羟化酶在中枢神经系统的正常功能中起着至关重要的作用。苯丙氨酸羟基酶的缺失导致苯丙酮尿症；酪氨酸羟基酶是合成神经递质多巴胺、去甲肾上腺素和肾上腺素所必需的；色氨酸羟基酶是合成神经递质5-羟色胺所必需的。

DESCRIPTION (provided by applicant): Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase make up the family of non-heme iron pterin-dependent aromatic amino acid hydroxylases. Each enzyme catalyzes the hydroxylation of the aromatic side chain of its substrate in a reaction critical for proper functioning of the central nervous system: phenylalanine metabolism, catecholamine biosynthesis, and serotonin biosynthesis. In addition to their physiological importance, these enzymes are unique in that they are the only nonheme iron enzymes whose physiological reaction is the hydroxylation of unactivated aromatic systems. The present proposal addresses several outstanding questions regarding the catalytic and regulatory mechanisms of these enzymes. The common catalytic mechanism will be studied using a combination of rapid reaction methods and spectroscopy, with the goal of characterizing individual iron species in catalysis. The role of protein dynamics in catalysis by tyrosine and phenylalanine hydroxylase will be studied by determining the effects of substrates on the kinetics of peptide bond exchange with solvent. The regulation of phenylalanine and tyrosine hydroxylase is critical to their proper function. The kinetics of peptide bond exchange will be used to evaluate a literature model for allosteric regulation of phenylalanine hydroxylase, while fluorescence methods will be used to evaluate a structural model for the regulation of tyrosine hydroxylase by phosphorylation. The active site iron atom in these enzymes is bound by a 2-Histidine-1-carboxylate facial triad. These two histidines in phenylalanine hydroxylase will be replaced with analogs and the effects on individual kinetic steps determined, using a combination of steady state kinetics, product partitioning, and kinetic isotope effects. The aromatic amino acid hydroxylases play critical roles in the proper functioning of the central nervous system. Loss of phenylalanine hydroxylase results in phenylketonuria; tyrosine hydroxylase is required to make the neurotransmitters dopamine, norepinephrine, and epinephrine; and tryptophan hydroxylase is required to synthesize the neurotransmitter serotonin.