THE BIOTRANSFORMATION OF ENDOBIOTICS BY SULFONATION

磺化在生物转化过程中起着至关重要的作用 内生生物和外生生物一样。磺化是一种主要的 众多结构和膜的平移后修饰 以及绝对必不可少的分泌物蛋白质 用于正常发育和维持健康。低硫的磺化反应 分子量化合物在激素作用中起着至关重要的作用， 储存和新陈代谢。通用的磺酸盐供体分子，3‘- 磷酸腺苷5‘-磷酸硫酸盐(PAPS)是由两种化合物合成的 催化反应，即ATP硫酰基酶和腺苷5‘- 磷酸硫酸盐(APS)激酶。与细菌、真菌、酵母和 这两种酶位于不同多肽链上的植物，在 在高等真核生物中，基因融合已经发生，这两个反应是 单一蛋白质(PAPS合酶)所固有的。我们克隆了人类， 豚鼠PAPS合成酶及其重组蛋白的初步研究 构建已经证明APS激酶活性驻留在 氨基末端结构域，而三磷酸腺苷硫酰基酶活性位于 COOH-蛋白质的末端结构域。我们目前正在进行 利用定点突变和重组构建物的研究 目的：1)确定高度保守的核苷酸结合的意义 APS激酶域中的基序；2)检测保守的FISP序列 存在于APS激酶结构域中，被认为是一种 APS磷酸化过程中的磷酸化中间体，形成PAPs； 3)确认存在基本的公共重叠序列 对于所建议的ATP硫酰基酶和APS激酶的功能 通过我们的初步研究；4)确定是否存在APS结合 重叠序列中的口袋；5)评估调控 COOH末端片段对APS定位蛋白激酶活性的影响 我们的动力学研究表明PAPS合成酶的NH2末端结构域 全长融合蛋白及其活性结构域的分析 通过重组技术生产。帕普斯是如此关键的事实 哺乳动物中的生物分子使其生产具有至关重要的作用。 反过来，这又清楚地将Paps Synthase置于战略地位。这个 更多关于这种耐人寻味的融合蛋白的了解 监管纸张的生产，人们就会更多地了解 磺化过程的进化、生物化学和生物学。这个 事实是硫化反应如此普遍，以至于它们涉及到 令人印象深刻的分子军团，既有大的(例如膜和 细胞外结构元素)和小的(例如，激素和 神经递质)，再加上PAPs是不可或缺的 和哺乳动物中通用的磺酸盐供体分子，它就不会 怀疑敲除PAPS合成酶基因可能是不合理的 事实证明是致命的。事实上，人类可遗传的致命性疾病 1型软骨生成有力地支持了这一结论。因此，可以说 正常运作的PAPS合成酶对生命本身是必需的 一点也不夸张。

Sulfonation is of fundamental importance in the biotransformation of endobiotics as well as exobiotics. Sulfonation plays a primary role in the post-translational modification of numerous structural and membrane constituents as well as secretary proteins that is absolutely essential for normal development and maintenance of health. The sulfonation of low molecular weight compounds plays a crucial role in hormone action, storage and metabolism. The universal sulfonate donor molecule, 3'- phosphoadenosine 5'-phosphosulfate (PAPS), is synthesized by two catalytic reactions, i.e., ATP sulfurylase and adenosine 5'- phosphosulfate (APS) kinase. In contrast to bacteria, fungi, yeast, and plants where the two enzymes are on separate polypeptide chains, in higher eukaryotes, gene fusion has occurred and the two reactions are intrinsic to a single protein (PAPS synthase). We have cloned human and guinea pig PAPS synthase and our preliminary studies with recombinant constructs have demonstrated that APS kinase activity resides within the NH2-terminal domain, while ATP sulfurylase activity is located in the COOH-terminal domain of the protein. We are currently carrying out studies employing site-directed mutagenesis and recombinant constructs to: 1) determine the significance of highly conserved nucleotide binding motifs within the APS kinase domain; 2) examine a conserved FISP sequence present in the APS kinase domain that is thought to function as a phosphorylated intermediate in the phosphorylation of APS to form PAPS; 3) confirm that there is a common overlapping sequence which is essential for the functionality of both ATP sulfurylase and APS kinase as suggested by our preliminary studies; 4) determine if there is an APS binding pocket within the overlapping sequence; 5) evaluate the regulatory influence of the COOH-terminal segment on APS kinase activity located in the NH2-terminal domain of PAPS synthase as suggested by our kinetic analyses of the full-length fusion protein and the active domains produced by recombinant techniques. The fact that PAPS is such a critical biological molecule in mammals makes its production of vital importance. This, in turn, clearly places PAPS synthase in a strategic position. The more that is understood about this intriguing fused protein that regulates the production of PAPS, the more that will be understood about the evolution, biochemistry and biology of the sulfonation process. The reality that sulfurylation reactions are so widespread, that they involve an impressive legion of molecules, both large (e.g. membrane and extracellular structural elements) and small (e.g. hormones and neurotransmitters), coupled with the fact that PAPS is the indispensable and universal sulfonate donor molecule in mammals, it would not be unreasonable to suspect that knocking out the PAPS synthase gene might prove to be lethal. In fact, the human heritable lethal disorder achondrogenesis type 1 cogently supports this conclusion. Thus, to say that a normally functioning PAPS synthase is mandatory for life itself would not be an overstatement.