MECHANISTIC STUDIES OF TYROSINE HYDROXYLASE

酪氨酸羟化酶催化酪氨酸的羟基化形成 二羟基苯丙氨酸，使用分子氧和四氢蝶呤作为 共底物 酪氨酸羟化酶作为催化剂的作用 儿茶酚胺生物合成中的限速步骤使酶 在个人健康中的核心作用。 儿茶酚胺失衡 许多疾病状态都涉及到高浓度。 高血压 是美国的一个主要健康问题;一些研究表明， 在高血压动物中发现了改变的儿茶酚胺代谢。 神经质 疾病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种情况下都会受到影响， 神经和精神疾病。 最后，一些药物， 法律的和非法的已经被证明直接影响的活动 体内酪氨酸羟化酶。 这里描述的研究目标 是为了确定这种重要酶的催化机制， 短期调控的机制和结构效应， 磷酸化 酪氨酸羟化酶的催化机制很差 明白 这种酶的每个活性部位含有一个亚铁原子 并且没有可见的发色团。 涉及亲电的机制 芳族取代、环氧化或羟基自由基攻击已经被 为这个或类似的系统。 为了区分这些 可能性：1）p- 取代的苯丙氨酸的取代基在 羟基化和非生产性四氢蝶呤氧化将被 测定2)同位素对p-和m-相对量的影响 由苯丙氨酸产生的酪氨酸将用于测试环氧化物 中间体3)来自非偶联周转的蝶呤产物将是 测定 探讨活性中心铁的作用：1）快速淬火 EPR光谱将用于确定活性位点铁 在催化过程中改变化合价。2)一氧化氮对血管内皮细胞的影响 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)圆二色谱 和低温磁性圆二色性将用于确定 铁原子的配位数和几何形状。 快速变化的速度生物合成的儿茶酚胺是 由酪氨酸羟化酶在几个丝氨酸上的磷酸化控制 残基 磷酸化-去磷酸化级联反应是迄今为止 快速和可逆地改变的活动的共同手段 内切酶 我们的目标是确定结构和催化变化 在磷酸化时发生的关键酶中。 以确定 磷酸化对催化作用的影响，1）对动力学的影响 并且丝氨酸磷酸化酶的pH依赖性将是 确定，和2）定点诱变将用于取代 Ser 4 O与其他氨基酸。

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.