Investigation of phenylalanine ammonia lyase and processing alpha glucosidase I

酶是高度进化的蛋白质，是大自然为特定目的精心设计的。通过充分了解它们的作用机制和结构，我们可以利用它们作为有效的生物合成工具，调节生化途径以获得所需的最终产物，或开发可以控制活性相关疾病状态的抑制剂。将研究来自两种酵母的两种不同的酶。在高等真核生物中，加工葡萄糖苷酶I （Glu I）对细胞功能至关重要，并有可能成为抗病毒、抗炎和抗癌药物的治疗靶点。酪氨酸/苯丙氨酸氨裂解酶（T/PAL）是一种对苯丙氨酸或酪氨酸具有广泛特异性的酶。这种特异性的基础尚不清楚。与酪氨酸的活性是非常理想的，因为产物对香豆酸具有很高的工业需求。更好地了解Glu I和T/PAL的机制和结构对于在健康和商业应用中控制或使用这些酶是必要的。在基础研究方面，这两种酶都提出了与酶的结构、功能和机制有关的有趣问题。本项目将开展以下工作：(1)对参与底物结合和催化的潜在关键氨基酸残基进行定点诱变，然后对突变体进行动力学和结构评价；(2)通过克隆和表达截断的基因产物来检测酶的功能域结构；(3)晶体学研究，进一步了解酶的结构和功能。这些实验将阐明Glu I和T/PAL的结构和机制的关键方面，并将使我们能够通过合理的抑制剂合成（Glu I）和设计酶来有效地生产工业上重要的对香豆酸（T/PAL）。

Enzymes are highly evolved proteins, exquisitely designed by nature for specific purposes. By fully understanding their mechanism of action and structure, we can exploit them as efficient biosynthetic tools, modulate biochemical pathways to obtain desired end-products, or develop inhibitor that can control activity-associated disease states. Two different enzymes from two yeasts will be investigated. Processing glucosidase I (Glu I) is critical to cell function in higher eukaryotes and has potential as a therapeutic target for anti-viral , anti-inflammatory, and anti-cancer drugs. Tyrosine/phenylalanine ammonia lyases (T/PAL) are enzymes with a wide range of specificty towards phenylalanine or tyrosine. The basis for this specificity is not understood. Activity with tyrosine is highly desirable, as the product, p-coumaric acid, has a high industrial demand. A better understanding of the mechanism and structure of Glu I and T/PAL is necessary to control or use these enzymes in health and commercial applications. In terms of basic research, both of these enzymes pose intriguing questions related to enzyme structure, function, and mechanism. This project will carry out the following: (1) site directed mutagenesis of potential key amino acid residues involved in substrate binding and catalysis , followed by kinetic and structural evaluation of the mutants; (2) examining the functional domain structure of the enzymes by cloning and expression of truncated gene products; (3) crystallographic studies to further our understanding of the enzyme structure/function. These experiments will clarify key aspects of the structure and mechanism of Glu I and T/PAL, and will allow us to exploit them through rational inhibitor synthesis (Glu I) and by designing enzymes to efficiently produce industrially important p-coumaric acid (T/PAL).