Mechanistic studies of Tyrosine Hydroxylase

酪氨酸羟化酶是一种含铁的、依赖羽翼蛋白的单加氧酶，在儿茶酚胺神经递质的生物合成中催化速率控制步骤，酪氨酸羟化形成二羟基苯丙氨酸。依赖羽翼素的单加氧酶是一组鲜为人知的酶。本研究的主要目的是了解酪氨酸羟化酶的催化机制。大鼠pc12酪氨酸羟化酶DNA已被克隆，并将在高效杆状病毒表达系统中表达，为详细的机制研究获得足够的纯材料。动力学机制将通过稳态动力学和同位素捕获来确定。快速反应技术将用于测量催化中间体的形成速率。几种非生理性氨基酸似乎作为酪氨酸羟化酶的底物；将对这些反应进行研究，以获得有关氧化物质身份的信息。这类酶，单加氧酶，催化生物化学中一些最困难的反应。因此，它们一直是大量研究的对象。与酪氨酸羟化酶相比，苯丙氨酸羟化酶的作用机制已经得到了很好的研究。它显示了一些显著的差异；苯丙氨酸似乎不是酪氨酸羟化酶的底物，尽管数据不一致；静息酶的氧化还原状态不同；酪氨酸羟化酶可以利用H2代替四氢蝶素。由于该研究与黄蛋白羟化酶和铁（11）单加氧酶具有共同的特征，因此该研究也可以为其他类单加氧酶的机制提供基本的见解。

Tyrosine hydroxylase, an iron-containing, pterin-dependent monoxygenase catalyze the rate-controlling step in the biosynthesis of catecholamine neurotransmitters, the hydroxlation of tyrosine to form dihydroxyphenylalanine. The pterin-dependent monooxygenases are a poorly understood group of enzymes. The primary goal of this research is to understand the catalytic mechanism of tyrosine hydroxylase. The DNA for rat PC 12 tyrosine hydroxylase has been cloned and will be expressed in a high efficiency baculovirus expression system in order to obtain enough pure material for detailed mechanistic studies. The kinetic mechanism will be determined using steady state kinetics and isotope trapping. Rapid reaction techniques will be used to measure the rates of formation of catalytic intermediates. Several nonphysiological amino acids appear to act as substrates for tyrosine hydroxylase; these reactions will be studies to gain information about the identity of the oxygenating species. This class of enzymes, the monooxygenases, catalyze some of the most difficult reactions in biochemistry. Consequently, they have been the objects of a great deal of study. The mechanism of phenylalanine hydroxylase has been well studied in contrast to tyrosine hydroxylase. It shows a number of significant differences; phenylalanine does not appear to be a substrate for tyrosine hydroxylase although the data is not consistent; the redox state of the resting enzymes are different; tyrosine hydroxylase can use H2 02 in place of tetrahydropterins. This study could also offer fundamental insights into the mechanism of other classes of monooxygenases since it has features in common with both flavoprotein hydroxylases and Fe(11) monooxygenases.