MECHANISTIC STUDIES OF TYROSINE HYDROXYLASE

酪氨酸羟基酶催化酪氨酸羟基化形成 二羟基苯丙氨酸，使用分子氧和四氢蝶呤作为 附属物。酪氨酸羟基酶对此的催化作用 儿茶酚胺生物合成中的限速步骤使酶 在一个人的健康中起着核心作用。儿茶酚胺的失衡 这一水平与许多疾病状态有关。高血压 是美国的一个主要健康问题；一些研究表明 在高血压动物体内发现儿茶酚胺代谢改变。神经质 混乱给社会带来了同样巨大的代价； 大脑中产生儿茶酚胺的区域在几个方面受到影响 神经学和精神病学疾病。最后，一些药物，都是 合法的和非法的都被证明直接影响到 体内的酪氨酸羟基酶。这里描述的研究目标 是为了确定这种重要酶的催化机制，以及 短期调控的机制和结构效应 磷酸化。 酪氨酸羟基酶的催化机理很差。 明白了。这种酶的每个活性部位含有一个铁原子。 并且没有可见的发色团。涉及亲电的机理 芳香族取代、环氧化或羟基自由基攻击 建议用于此系统或类似系统。要区分这两个 可能性：1)电子给电子能力的影响。 取代苯丙氨酸的取代基对分配的影响 羟化和无效的四氢蝶呤氧化将 下定决心。2)同位素对p-和m-的相对含量的影响 由苯丙氨酸产生的酪氨酸将被用来测试环氧化物 中级的。3)来自非偶联营业额的蝶呤产品将 下定决心。探讨活性部位铁的作用：1)快速淬火 电子顺磁共振波谱将被用来确定活性位是否铁 在催化过程中改变价态。2)一氧化氮对大鼠心脏功能的影响 酶的Fe(II)形式的EPR信号将在 底物和抑制剂的存在和不存在。3)圆二向色性 并将利用低温磁性圆二色谱来确定 铁原子的配位数和几何构型。 儿茶酚胺生物合成速率的快速变化是 由几个丝氨酸基酪氨酸羟基酶的磷酸化控制 残留物。到目前为止，磷酸化-去磷酸化级联反应是最 快速和可逆地修改活动的常用手段 酵素。我们的目标是确定结构和催化变化 在这种关键的酶中，在磷酸化时发生。要确定 磷酸化对催化的影响：1)对动力学的影响 Ser4O上磷酸化酶的pH依赖性为 确定的，以及2)将使用定点突变来取代 Ser4O与其他氨基酸结合。

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine, using molecular oxygen and a tetrahydropterin as cosubstrates. The role of tyrosine hydroxylase as the catalyst for this rate-limiting step in catecholamine biosynthesis gives the enzyme a central role in the health of an individual. Imbalances in catecholamine levels have been implicated in a number of disease states. Hypertension is a major health problem in the United States; a number of studies have found altered catecholamine metabolism in hypertensive animals. Neurotic disorders impose an equally large cost upon society; the catecholamine-producing areas of the brain are affected in several neurologic and psychiatric diseases. Finally, a number of drugs, both legal and illegal have been shown to directly effect the activity of tyrosine hydroxylase in vivo. The goals of the research described here are to determine the catalytic mechanism of this important enzyme and the mechanistic and structural effects of short term regulation by phosphorylation. The catalytic mechanism of tyrosine hydroxylase is very poorly understood. The enzyme contains a single ferrous atom per active site and has no visible chromophore. Mechanisms involving electrophilic aromatic substitution, epoxidation, or hydroxyl radical attack have been proposed for this or similar systems. To distinguish among these possibilities: 1) The effect of the electron donating ability of p- substituents of substituted phenylalanines on the partitioning between hydroxylation and unproductive tetrahydropterin oxidation will be determined. 2) Isotope effects on the relative amounts of p- and m- tyrosine produced from phenylalanine will be used to test for an epoxide intermediate. 3) The pterin product from uncoupled turnover will be determined. To probe the role of the active site iron: 1) Rapid quench epr spectroscopy will be used to determine if the active site iron changes valency during catalysis. 2) The effect of nitric oxide on the epr signal of the Fe(II) form of the enzyme will be determined in the presence and absence of substrates and inhibitors. 3) Circular dichroism and low temperature magnetic circular dichroism will be used to determine the coordination number and geometry of the iron atom. Rapid changes in the rate of biosynthesis of catecholamines are controlled by phosphorylation of tyrosine hydroxylase at several seryl residues. Phosphorylation-dephosphorylation cascades are by far the most common means of rapidly and reversibly modifying the activities of enzymes. Our goal is to determine the structural and catalytic changes in this critical enzyme which occur upon phosphorylation. To determine the effect of phosphorylation on catalysis, 1) the effect on the kinetics and the pH dependence of enzyme of phosphorylation at Ser4O will be determined, and 2) site directed mutagenesis will be used to replace Ser4O with other amino acids.