MECHANISMS OF ENZYMIC AND HYDRIDE TRANSFERS

这个项目的总体目标是了解有效的酶催化从弱酸性碳和氧中心进行氢转移的结构基础。我们选择了几个有代表性的制度进行研究。对于质子转移催化：磷酸葡萄糖异构酶(PGI)，它首先催化质子转移到氧(开环)，然后利用酸/碱催化的碳间质子转移来异构化磷酸糖，以及D-半乳糖转位酶(GALM)，它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)，它通过金属介导的1，2-氢化物转移在碳中心之间转移氢，并催化氧原子之间的质子转移，即糖环打开；以及肌苷5‘-单磷酸脱氢酶(IMPDH)，它使用NAD+作为氢化物受体，将肌苷转化为黄苷。为了测试各种残基在这些酶的作用机制中的可能作用，我们将结合使用定点突变和X射线结晶学。除了了解活性中心残基的确切化学和结构作用外，这些研究还将提供反应途径的观点，并定性和半定量地评估以下因素对质子和氢化物转移催化的贡献：总酸/总碱催化；亲电催化；到过渡态的短而强的氢键；带电物种的静电稳定，结合水分子的参与，以及底物/辅因子菌株。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性疾病中发生突变。此外，PGI以某种未知的机制从兼职的细胞中分泌出来，作为一种强大的细胞因子和肿瘤细胞减数因子。XYI在食品生产中很重要，也被用于生物技术。Ga1M是半乳糖利用的Leloir途径的一部分，有许多已知的半乳糖代谢紊乱。IMPDH催化GMT生物合成的速率决定步骤，是免疫抑制、抗癌、抗病毒和抗微生物药物的靶点。

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.