MECHANISMS OF ENZYMATIC REACTIONS

酶促反应机制

• 批准号: 6385438
• 负责人: PERRY A. FREY
• 金额: $ 36.02万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-04-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385438
• 关键词: Escherichia coli; UTP hexose 1 phosphate uridylyltransferase; X ray crystallography; active sites; bioenergetics; carbohydrate metabolism; charge transfer complex; cofactor; conformation; enzyme mechanism; enzyme structure; enzyme substrate complex; galactokinase; galactose; hydro lyase; isomerase; nicotinamide adenine dinucleotide; site directed mutagenesis; stereochemistry

The long term objective is to elucidate the mechanisms by which binding energy in enzyme-substrate complexes facilitates enzymatic catalysis. A global approach will be adopted in coordinated studies of four enzymes that participate in carbohydrate metabolism and phosphotransfer, UDP- galactose 4-epimerase (Ga1E), galactose-1-phosphate uridlyltransferase (Ga1T), galactokinase (Ga1K), dTDP-glucose 4,6-dehydratase (dehydratase) and human Fhit, a putative tumor suppressor. Chemical, kinetic, spectroscopic, mutagenic, and crystallographic methods will be employed. Galactose metabolism is essential in all living cells and presents fundamental questions bearing on binding energy and catalysis. Cells must break galactose down for use as a fuel to produce galactose for glycoconjugates. One manifestation of the importance of galactose metabolism is the metabolic defect underlying galactosemia, in which galactose metabolism is impaired by defects in GalT. Research on three enzymes of galactose metabolism will be emphasized. The enzymes from E. coli will be studied as models for the mammalian enzymes, which are homologous. The principle objective in studies of Ga1E will be to describe the molecular basis for the enhancement of the chemical reactivity of the niacin- co-enzyme NAD+ by the use of binding energy between the UDP-moiety of substrates and the enzyme. Another Ga1E objective is to elucidate the mechanism of general acid/base catalysis, which appears to be carried out by tyrosine 149 and serine 124. The structural basis for a charge-transfer interaction between NAD+ and the enzyme will be determined. Research on dehydratase will be compared and contrast its biological mechanism with that of Ga1E. The objectives for research on Ga1T include the elucidation of how binding energy is used to stabilize the uridylyl-enzyme intermediate, how metal ions stabilize the active conformation, and how specific galactosemia mutations undermine the enzymatic activity. The standard free energy for the formation of the uridylyl-enzyme in site-directed mutant forms will be compared with wild-type enzyme to investigate binding interactions that stabilize the intermediate. Research on Fhit is directed toward elucidating its structural and mechanistic relationships with GalT. Research on the GalK will be initiated to determine its chemical mechanism and structure. The stereochemical course of phosphotransfer by GalK will be unmasked to determine whether a single or double- displacement mechanism is in full operation. Crystallization trials will be pursued with the objective of determining the first structured in the Ga1K family of sugar phosphotransferases.

长期目标是阐明酶-底物复合物中的结合能促进酶催化的机制。将采用全球方法对参与碳水化合物代谢和磷酸转移的四种酶进行协调研究，即UDP-半乳糖4-差向异构酶（Ga1E）、半乳糖-1-磷酸尿苷酰转移酶（Ga1T）、半乳糖激酶（Ga1K）、dTDP-葡萄糖4、 6-脱水酶（脱水酶）和人 Fhit（一种假定的肿瘤抑制因子）。将采用化学、动力学、光谱、诱变和晶体学方法。半乳糖代谢对于所有活细胞至关重要，并提出了与结合能和催化有关的基本问题。细胞必须分解半乳糖作为燃料来生产糖复合物的半乳糖。半乳糖代谢重要性的表现之一是半乳糖血症的代谢缺陷，其中半乳糖代谢因 GalT 缺陷而受损。重点研究半乳糖代谢的三种酶。来自大肠杆菌的酶将作为同源的哺乳动物酶的模型进行研究。 Ga1E 研究的主要目标是描述通过利用底物的 UDP 部分与酶之间的结合能来增强烟酸辅酶 NAD+ 化学反应性的分子基础。 Ga1E 的另一个目标是阐明一般酸/碱催化的机制，该机制似乎是由酪氨酸 149 和丝氨酸 124 进行的。将确定 NAD+ 和酶之间电荷转移相互作用的结构基础。脱水酶的研究将其生物学机制与Ga1E进行比较和对比。 Ga1T 研究的目标包括阐明如何使用结合能来稳定尿嘧啶酶中间体、金属离子如何稳定活性构象以及特定的半乳糖突变如何破坏酶活性。将定点突变形式的尿嘧啶酶形成的标准自由能与野生型酶进行比较，以研究稳定中间体的结合相互作用。对 Fhit 的研究旨在阐明其与 GalT 的结构和机制关系。将启动对GalK的研究以确定其化学机制和结构。 GalK 磷酸转移的立体化学过程将被揭示，以确定单位移机制还是双位移机制是否处于完全运行状态。将进行结晶试验，目的是确定 Ga1K 糖磷酸转移酶家族中的第一个结构。