EUKARYOTIC HYALURONAN SYNTHASES

结构元件，黏附的中介物和细胞外信号 分子。透明质酸多糖的含量和质量 调节发育过程中的细胞行为，细胞间黏附和 识别、运动和新生血管。的长期目标是 本研究旨在阐明其结构和作用机制。 腐植酸合成酶在植物体内产生透明质酸的作用 人类。脊椎动物体内HA的生物合成已被研究 几十年来收效甚微，直到1996年，一个真核HA家族 鉴定了合成酶。这些酶是新颖而有趣的。 糖基转移酶，因为它们转移两个不同的糖基 在聚合过程中，几乎所有其他酶只转移一种糖。 脊椎动物透明质酸生物合成的分子和生化细节 都是缺乏的。因此，这个五年项目期的主要目标是 是对非洲爪哇和人类的几种HA合成酶进行鉴定。这个 研究人员已生产出具有功能的重组非洲爪哇HA合成酶 无背景体系中的酶。这个项目的具体目标是 1)分析真核细胞中HA合成酶的动力学。 经典的生化研究将揭示酶以何种方式 聚合其UDP-糖前体。2)从结构上描述 真核细胞的透明质酸合成酶。辐射灭活与质谱学 将用于定义功能单元和单体尺寸， 分别进行了分析。3)确定HA的前体结合部位 合成酶。将利用光亲和力和化学标记研究 与UDP-糖底物相互作用的酶的OT标记区。 4)测定HA合成酶的功能残基。残留物 在HA合成酶中保守的基因将通过定点改变 突变；特别是某些半胱氨酸残基将因 它们在催化和二硫键形成中的潜在作用。 目标3中确定的基本残留物也将得到确认 诱变研究。酶学和结构信息可以 允许生成描述催化机理的工作模型 这些酶中。

Structural element, a mediator of adhesion, and an extracellular signaling molecule. The quantity and the qualities of the HA polysaccharide modulate cell behavior during development, intercellular adhesion and recognition, locomotion, and neovascularization. The long-term goal of this investigation is to elucidate the structure and the mechanism of action of the HA synthase enzymes responsible for production of HA in humans. The biosynthesis of HA in vertebrates has been studied for decades with little success until 1996, when a family of eukaryotic HA synthases was identified. These enzymes are novel and interesting glycosyltransferases because they transfer two different sugar groups during polymerization; almost all other enzymes transfer only one sugar. The molecular and biochemical details of HA biosynthesis in vertebrates are lacking. Therefore, the major goal of this five-year project period is to characterize several HA synthases from Xenopus and humans. The investigators have produced functional recombinant xenopus HA synthase enzyme in a background-free system. The specific aims of this project are; 1) To analyze the kinetics of the eukaryotic HA synthases. Classical biochemical studies will reveal the manner in which the enzyme polymerizes its UDP-sugar precursors. 2) To characterize structurally the eukaryotic HA synthases. Radiation inactivation and mass spectrometry will be used to define the functional unit and monomer sizes, respectively. 3) To identify the precursor binding sites of the HA synthases. photoaffinity and chemical labeling studies will be utilized ot tag regions of the enzyme that interact with the UDP-sugar substrates. 4) To determine the functional residues of the HA synthase. The residues that are conserved among HA synthases will be altered by site-directed mutagenesis; in particular, certain cysteine residues will be mutated due to their potential roles in catalysis and in disulfide bond formation. The essential residues identified in aim 3 will also be confirmed by mutagenesis studies. The enzymological and structural information may allow the generation of a working model describing the catalytic mechanism of these enzymes.