SULFATE ADENYLATION-BIOCHEMISTRY & ENZYMOLOGY

描述(改编自申请人的摘要)：三磷酸腺苷硫酰酶，来自大肠杆菌， 催化和构象偶联GTP水解的自由能和 活化硫酸盐合成(反应1和反应2)。酶是唯一的例子 结合两种化学物质的自由能的GTP酶/靶复合体 反应。在催化循环中，分子间的变构作用 活跃的网站驱动结构变化，控制着 个人反应。这些链接事件导致相互依赖， 固定了反应的化学计量比，并耦合了它们的自由能。这个 拟议的研究集中在这些联系事件上，并将建立 GTP酶/靶蛋白中变构相互作用的范例 络合物和自由能的构象耦合。 ATP+SO(4)&&gt；APS+PPI(1) GTP+H(2)O&lt；-&gt；GDP+PI+H(+)(2) 在硫酸盐活化途径的第二步也是最后一步， 三磷酸腺苷的伽马-磷酰基转移到APS的3‘-羟基上形成 PAPS(反应3)。 ATP+APS；-&>PAPS+ADP(3) PAPS是唯一已知的代谢中的硫基供体。硫基转移， 就像磷酸转移一样，被细胞广泛地用来调节 代谢物活性。在人类中，整个硫酸盐激活途径是 含有一种单一的酶，即PAPS合成酶。这个单体73 kDa 多肽催化反应1和反应3及其之间的通道 活动站点。人PAPS合成酶已被表达和纯化到 首席调查员实验室中的同质性。的结构 通灵系统是罕见的，它揭示了令人惊叹的架构 这些酶用来在它们的活性部位之间分流底物。PAPS 合成酶有望成为这些系统中的一个。PAPS合成酶的晶体 已经得到了衍射率为2.8埃的结果。现有的范式 表明通道将需要广泛的变构相互作用 PAPS合成酶的活性部位。莱伊博士将启动一项结构性和 对这个令人着迷的系统的功能描述。

DESCRIPTION(adapted from applicant's abstract): ATP sulfurylase, from E. coli, catalyzes and conformationally couples the free energies of GTP hydrolysis and activated sulfate synthesis (reactions 1 and 2). The enzyme is the only example of a GTPase/target complex that couples the free energy of two chemical reactions. During the catalytic cycle, allosteric interactions between the active sites drive structural changes that control the progression of the individual reactions. These linking events result in an interdependence that fixes the stoichiometry of the reactions, and couples their free energies. The proposed studies are focused on these linking events and will establish paradigms for the allosteric interactions that occur in GTPase/target complexes, and the conformational coupling of free energy. ATP + SO(4) <-> APS + PPi (1) GTP + H(2)O <-> GDP + Pi + H(+) (2) In the second and final step of the sulfate activation pathway, the gamma-phosphoryl group of ATP is transferred to the 3'-hydroxyl of APS to form PAPS (reaction 3). ATP + APS <-> PAPS + ADP (3) PAPS is the only known sulfuryl group donor in metabolism. Sulfuryl transfer, much like phosphoryl transfer, is used extensively by the cell to regulate metabolite activity. In humans, the entire sulfate activating pathway is contained in a single enzyme, PAPS synthetase. This monomeric 73 kDa polypeptide catalyzes reactions 1 and 3 and channels APS between its active-sites. The human PAPS synthetase has been expressed and purified to homogeneity in the principal investigator's laboratory. Structures of channeling systems are rare and have revealed the amazing architecture that these enzymes use to shunt substrates between their active sites. PAPS synthetase promises to be one of those systems. Crystals of PAPS synthetase have been obtained that diffract to 2.8 angstroms. The existing paradigms suggest that channeling will require extensive allosteric interactions between active sites of PAPS synthetase. Dr. Leyh will initiate a structural and functional characterization of this fascinating system.