Enzymology of Inorganic Sulfate Metabolism

大多数细菌、酵母菌、真菌、藻类和高等植物都利用 无机硫酸盐作为其生物合成的唯一硫源 半胱氨酸，蛋氨酸，所有还原有机含硫 辅酶（生物素、硫胺素、辅酶A、硫辛酸等），和 各种结构成分（植物硫脂，硫酸化 多糖等）。 研究的总体目标是 建立催化机制，调节特性， 结构-功能关系的酶催化的 真菌同化无机硫酸盐的最早阶段。 因此，在本发明中， 对酶（a）ATP硫酸化酶，（B）APS激酶，和 （c）第（1）款 “PAPS还原酶”，其依次催化（a） 腺苷-5 '-磷酸硫酸盐（APS）从SO 42-和ATP中分离，（B） 由3 '-磷酸腺苷-5'-磷酸硫酸盐（PAPS）形成 APS和ATP，以及（c）NADPH和硫氧还蛋白依赖性还原 PAPS与游离或结合的亚硫酸盐的结合。 “PAPS还原酶”实际上是一种 从未被纯化至均匀的多组分体系 的动力学特征。 大多数实验将是 用从嗜温真菌中纯化的酶进行， 产黄青霉 平行研究将在 酶从嗜热菌，杜蓬青霉菌与 目的是确定负责的结构特征 这种生物的蛋白质具有非凡的热稳定性。 酶动力学，平衡结合，蛋白质化学，和 将使用分子生物学方法。

Most bacteria, yeasts, fungi, algae, and higher plants use inorganic sulfate as their sole sulfur source for the biosynthesis of cysteine, methionine, all reduced organic sulfur-containing coenzymes (biotin, thiamin, coenzyme A, lipoate, etc.), and a variety of structural components (plant sulfolipid, sulfated polysaccharides, etc.). The overall objective of the research is to establish the catalytic mechanism, regulatory properties, and structure-function relationships of the enzymes which catalyze the earliest steps of inorganic sulfate assimilation by fungi. Thus, a study of the enzymes (a) ATP sulfurylase, (b) APS kinase, and (c) "PAPS reductase" which catalzye, in order, (a) the formation of adenosine-5'- phosphosulfate (APS) from SO42- and ATP, (b) the formation of 3'-phosphoadenosine- 5'- phosphosulfate (PAPS) from APS and ATP, and (c) the NADPH and thioredoxin-dependent reduction of PAPS to free or bound sulfite. "PAPS reductase" is actually a multicomponent system which has never been purified to homogeneity of kinetically characterized. Most of the experiments will be performed with enzymes purified from the mesophilic fungus, Penicillium chrysogenum. Parallel studies will be performed on the enzymes from the thermophile, Penicillium duponti with the objective of identifying the structural features responsible for the remarkable heat stability of proteins from this organism. Enzyme kinetics, equilibrium binding, protein chemistry, and molecular biology methods will be used.