MECHANISMS OF ENZYMATIC REACTIONS

酶促反应机制

• 批准号: 2444531
• 负责人: PERRY A. FREY
• 金额: $ 29.78万
• 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 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444531
• 关键词: Escherichia coli; UTP hexose 1 phosphate uridylyltransferase; X ray crystallography; active sites; charge transfer complex; chemical binding; chemical reaction; enzyme mechanism; galactose; gene mutation; hydrolysis; isomerase; molecular site; site directed mutagenesis; uridine diphosphate

DESCRIPTION: The long term specific aim is to elucidate at the atomic level the mechanisms by which binding energy from enzyme-substrate interactions is utilized to facilitate enzymatic catalysis. This objective will be pursued through a global approach utilizing the methods of chemistry, kinetics, spectroscopy, site-directed mutagenesis, and X-ray crystallography. The metabolism of galactose presents fundamental questions bearing on this issue; moreover, galactose metabolism is essential in all living cells, which must break galactose down for use as a fuel and also produce galactose for the biosynthesis of glycoconjugates. One manifestation of the importance of galactose metabolism is the metabolic defect underlying galactosemia, in which galactose metabolism is impaired by structural defects in hexose-1-P uridylyltransferase. In the current grant period, research on UDP- galactose 4-empimerase (epimerase), hexose-1-P uridylyltransferase (uridylyltransferase), and galactose mutarotase (mutarotase) will be emphasized. The enzymes from E. coli will be studied as models for the mammalian enzymes, which are homologous. The specific objectives for epimerase include the elucidation of the structural and kinetic basis for the effect of binding interactions between the UDP-moiety of substrates and the enzyme in enhancing the chemical reactivity of NAD+, the coenzyme that facilitates epimerization at glycosyl C-4 of substrates. Another epimerase objective is to apply structural tests to the prevailing hypothesis accounting for nonstereospecific hydrogen transfer. The glycosyl binding subsite and the catalytic general base/acid within this site will be identified. The structural basis for a charge-transfer interaction between NAD+ and the enzyme will be elucidated. The objectives for research on uridylyltransferase include the structural characterization of the active site in both the resting enzyme and the covalent uridylyl-enzyme will be measured and compared with those for model uridylyl imidazolates to determine the effect of enzymatic binding ont he stability of the intermediate. The glycosyl binding subsite will be identified and characterized. The roles of Zn and Fe ions bound to this enzyme will also be investigated. The current structure reveals the identities of the ligands to these metals to be the side chains of four histidines, two cysteines, and one glutamate. The human enzyme is homologous but lacks one histidine corresponding to His115 that coordinates to Zn in the E. coli enzyme. The human sequence of SerAsp will be introduced into the E. coli enzyme and the structural and kinetic consequences evaluated. Research on the galactose mutarotase will be initiated to determine its chemical mechanism and structure. The enzyme will be purified and subjected to crystallization trials. No mutarotase mechanism has been established and four possible mechanisms will be tested with the objective of eliminating all but one. This one will then be characterized by the application of structural, chemical, kinetic, and spectroscopic techniques.

描述：长期特定的目的是阐明原子 将结合能量的能量从酶 - 底物中升级 相互作用用于促进酶促催化。 这 将通过使用全球方法来实现目标 化学，动力学，光谱，定点诱变的方法， 和X射线晶体学。 半乳糖的代谢 关于这个问题的基本问题； 此外，半乳糖 代谢在所有活细胞中都是必不可少的，必须破坏银乳糖 向下用作燃料，还为生物合成产生半乳糖 糖缀合物。 半乳糖重要性的一种表现 新陈代谢是半乳糖血症的代谢缺陷，其中 半乳糖代谢受到己糖1-P中的结构缺陷的损害 尿液转移酶。 在当前的赠款期，对UDP-的研究 半乳糖4- EMPIMERASE（EPIMERASE），己糖-1-P尿苷转移酶 （尿液转移酶）和半乳糖素蛋白介酶（Mutarotase）将是 强调。 将研究来自大肠杆菌的酶作为模型 同源的哺乳动物酶。 特定目标 分配酶包括阐明结构和动力学基础的基础 底物的UDP运动之间的结合相互作用的影响 以及增强NAD+化学反应性的酶， 辅助辅酶，可促进底物糖基C-4的表达。 另一个分配酶的目标是将结构测试应用于 非特异性氢的普遍假设 转移。 糖基结合亚铁矿和催化一般 将确定该站点内的基础/酸。 结构性基础 NAD+和酶之间的电荷转移相互作用将是 阐明。 尿路转移酶的研究目标包括 两个静止位置的活性位点的结构表征 将测量并比较酶和共价尿苷 - 酶 与模型尿液咪唑酸盐的模型一起确定的效果 酶结合的稳定性。 糖基 将确定和表征结合子站点。 Zn的角色 与该酶约束的Fe离子也将进行研究。 电流 结构揭示了配体对这些金属的身份 四个组氨酸，两个半胱氨酸和一个谷氨酸的侧链。 人酶是同源的，但缺乏对应于 His115在大肠杆菌酶中与Zn协调。 人类序列 SERASP将被引入大肠杆菌和结构 和动力学后果。 银河系的研究 将启动mutarotase以确定其化学机制和 结构。 酶将被纯化并进行结晶 试验。 尚未建立Mutarotase机制，并且可能有四种 机制将进行测试，目的是消除除一个人以外的全部。 然后，这将以结构性应用的特征 化学，动力学和光谱技术。