MECHANISMS OF ENZYMIC AND HYDRIDE TRANSFERS

酶和氢化物转移的机制

• 批准号: 6179634
• 负责人: GREGORY A PETSKO
• 金额: $ 22.49万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-16 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179634
• 关键词: X ray crystallography; acidity /alkalinity; chemical binding; chemical kinetics; enzyme mechanism; enzyme structure; enzyme substrate; glucose 6 phosphate isomerase; hydrogen bond; ionic bond; isomerase; oxidoreductase; protein engineering; purine nucleotides; site directed mutagenesis; xylose

The overall aim of this project is to understand the structural basis for efficient enzymic catalysis of hydrogen transfer from weakly acidic carbon and oxygen centers. We have selected several representative systems for study. For proton transfer catalysis: phosphoglucose isomerase (PGI), which first catalyzes proton transfer to oxygen (ring-opening) and then uses acid/base catalyzed proton transfer between carbons to isomerize a sugar phosphate, and D-galactose mutarotase (GalM), which catalyses ring-opening and proton transfer between oxygens on a sugar substrate that is not phosphorylated. For hydride transfer: xylose isomerase (XyI), which transfers hydrogen between carbon centers via metal-mediated 1,2-hydride shift and also catalyzes proton transfer between oxygens, i.e., sugar ring opening; and inosine 5'-monophosphate dehydrogenase (IMPDH), which uses NAD+ as a hydride acceptor in the transformation of inosine to xanthosine. To test the putative roles of various residues in the mechanisms of these enzymes, we will use a combination of site-directed mutagenesis and X-ray crystallography. In addition to an understanding of the precise chemical and structural roles of the residues in the active sites, these studies will provide views of the reaction pathways and a qualitative and semiquantitative assessment of the contribution to proton and hydride transfer catalysis of factors such as: general-acid/general-base catalysis; electrophilic catalysis; short, strong hydrogen bonds to the transition state; electrostatic stabilization of charged species, participation of bound water molecules, and substrate/cofactor strain. Understanding the mechanisms of action of these enzymes also has important consequences for human health. PGI is mutated in a severe hereditary metabolic disease. In addition, PGI by some unknown mechanism is secreted from the cell where it moonlights as a potent cytokine and tumor cell meidator. XyI is important in food production and is also being used in biotechnology. Ga1M is part of the Leloir pathway for the utilization of galactose and there are many known galactose metabolic disorders. IMPDH catalyzes the rate-determining step in GMT biosynthesis and is a target for immunosuppressive, anticancer, antiviral and antimicrobial drugs.

本项目的总体目标是了解从弱酸性碳和氧中心进行氢转移的有效酶催化的结构基础。 我们选择了几个有代表性的系统进行研究。 对于质子转移催化：磷酸葡萄糖异构酶（PGI），其首先催化质子转移到氧（开环），然后使用酸/碱催化的碳之间的质子转移来异构化糖磷酸，和D-半乳糖变旋酶（GalM），其催化未磷酸化的糖底物上的氧之间的开环和质子转移。 对于氢化物转移：木糖异构酶（XyI），其通过金属介导的1，2-氢化物移位在碳中心之间转移氢，并且还催化氧之间的质子转移，即，糖环开环;和肌苷5’-单磷酸脱氢酶（IMPDH），其在肌苷转化为黄苷中使用NAD+作为氢化物受体。为了测试这些酶的机制中的各种残基的假定作用，我们将使用定点诱变和X射线晶体学的组合。 除了对活性位点中残基的精确化学和结构作用的理解之外，这些研究还将提供反应途径的观点以及对质子和氢化物转移催化的贡献的定性和半定量评估，这些因素包括：一般酸/一般碱催化;亲电催化;与过渡态的短而强的氢键;带电物质的静电稳定、结合水分子的参与和底物/辅因子应变。了解这些酶的作用机制对人类健康也有重要影响。 PGI在一种严重的遗传性代谢疾病中发生突变。 此外，PGI通过一些未知的机制从细胞中分泌，在那里它兼职作为有效的细胞因子和肿瘤细胞减数分裂剂。 XyI在食品生产中很重要，也被用于生物技术。 Ga 1 M是利用半乳糖的Leloir途径的一部分，并且存在许多已知的半乳糖代谢障碍。 IMPDH催化GMT生物合成中的速率决定步骤，并且是免疫抑制、抗癌、抗病毒和抗微生物药物的靶标。