Enzymology of Inorganic Sulfate Metabolism

大多数细菌、酵母、真菌、藻类和高等植物可以使用无机硫酸盐作为其唯一的硫源，用于半胱氨酸、蛋氨酸和各种有机含硫辅酶的生物合成。本研究的目的是建立催化硫酸盐同化早期步骤的酶的结构-功能关系。研究了ATP硫酰化酶、APS激酶和PAPS还原酶。前两个催化两步ATP依赖的S042“激活”，形成3'-磷酸腺苷- 5' -硫酸磷酸（PAPS）。最后一个命名的酶（实际上是一个多组分系统）催化NADPH和硫氧还蛋白依赖的PAPS还原为无机亚硫酸盐。具体目标包括(a)确定动力学和化学机制，(b)确定活性位点的氨基酰基残基，以及(c)探索两种硫酸盐激活酶形成“PAPS合成酶”复合物的潜在相互作用。实验将使用从丝状真菌青霉菌和菠菜叶中纯化的酶进行。平行研究将在P. duponti（一种嗜热菌）酶上进行，目的是确定嗜热菌酶具有显著热稳定性的结构特征。酶动力学，蛋白质化学和分子生物学方法将被使用。在植物和微生物中，无机硫酸盐通过复杂的途径被还原到适合与含硫氨基酸和其他因子结合的状态。研究者采用一种复杂的数学方法来研究这些酶。他希望分析它们进行相关反应的化学机制。

Most bacteria, yeast, fungi, algae and higher plants can use inorganic sulfate as their sole source of sulfur for the biosynthesis of cysteine, methionine, and a variety of organic sulfur-containing coenzymes. The objective of this research is to establish the structure-function relationships of the enzymes which catalyze the early steps of sulfate assimilation. The enzymes ATP Sulfurylase, APS kinase, and PAPS reductase will be studied. The first two catalyze the two-step ATP- dependent "activation" of S042- forming 3'- phosphoadenosine- 5' -phosphosulfate (PAPS). The last named enzyme (actually a multicomponent system) catalyzes the NADPH- and thioredoxin- dependent reduction of PAPS to inorganic sulfite. Specific aims include (a) determining the kinetic and chemical mechanisms, (b) identifying aminoacyl residues at the active site, and (c) exploring the potential interaction of the two sulfate activating enzymes to form a "PAPS synthetase" complex. The experiments will be conducted with enzymes purified from the filamentous fungus Penicillium chrysogenum and from spinach leaf. Parallel studies will be performed on the enzymes from P. duponti (a thermophile) with the objective of identifying the structural features responsible for the remarkable heat stability of thermophile enzymes. Enzyme kinetics, protein chemistry, and molecular biology methods will be used. Inorganic sulfate is reduced to a state suitable for incorporation into sulfur containing amino acids and other factors by a complex pathway in plants and microorganizms. The investigator employs a sophisticated mathematical approach to the study of these enzymes. He wishes to analyze the chemical mechanisms by which they carry out the reactions involved.