Structure-Function Studies on the Sulfate Activating Enzymes

9904003Segel，Fisher这项研究考察了“硫酸盐激活酶”、ATP硫酰基酶和APS激酶催化和调节特性背后的结构特征。这些酶依次催化无机硫酸盐同化的前两个反应。研究了产黄青霉的同源六聚体三磷酸腺苷硫化酶，该酶被PAPs变构抑制。这种反馈过程是丝状真菌所特有的，很可能反映了这样一个事实，即PAPS是真菌中的主要分支点代谢物，而在其他生物中不是。真菌三磷酸腺苷硫酰基酶的变构结构域位于多肽链的C末端，与APSkinase有很高的同源性。实验旨在确定(A)变构结构域的三维结构与真APS激酶的三维结构之间的关系，以及(B)变构效应诱导的转变的结构性质。为了建立(A)硫代核苷酸的高度专一性，(B)底物的强制有序结合(是动力学的还是基于结构的？)，以及(C)亚单位结合和活性部位的形成之间的关系的结构基础，我们还研究了同源二聚体P.chrysgenum APS激酶。含有硫的关键生物分子包括蛋白质氨基酸、半胱氨酸和蛋氨酸，以及维生素泛酸、生物素和硫胺素。地球表面(土壤和海洋等)上的大部分硫。以无机硫酸盐的形式存在。本项目的重点是催化无机硫酸盐进入生物分子的前两个反应的ATP硫酰基酶和APS激酶的结构和功能。这些酶将从丝状真菌黄青霉(Penicillium Chrysgenum)中提纯出来(它会产生另一种重要的硫化合物青霉素)。

9904003Segel, Fisher This research examines the structural features underlying the catalyticand regulatory properties of the "sulfate activating enzymes", ATPsulfurylase and APS kinase. These enzymes catalyze, in order, the first tworeactions in the assimilation of inorganic sulfate. The investigation focuses on the homo-hexameric ATP sulfurylase of Penicillium chrysogenum,which is allosterically inhibited by PAPS. This feedback process is uniqueto filamentous fungi, very likely reflecting the fact that PAPS is a majorbranch point metabolite in fungi, but not in other organisms. Theallosteric domain of fungal ATP sulfurylase is located in the C-terminalregion of the polypeptide chain and has a high degree of homology with APSkinase. Experiments are designed to determine (a) the relationshipbetween the three dimensional structure of the allosteric domain and thatof true APS kinase and (b) the structural nature of the transition inducedby the allosteric effector. P. chrysogenum APS kinase, a homodimer, isalso be studied in order to establish the structural basis of (a) the highspecificity for sulfonucleotides, (b) the compulsory ordered binding ofsubstrates (is it kinetic or structurally-based?), and (c) the relationshipbetween subunit association and formation of the active site.Sulfur is one of the six major elements that compose living cells. Keybiological molecules that contain sulfur include the protein amino acids,cysteine and methionine, and the vitamins pantothenic acid, biotin, andthiamin. Most of the sulfur on the Earth's surface (in the soil and oceans,etc.) is present as inorganic sulfate. This project focuses on the structures and functions of ATP sulfurylase and APS kinase, enzymes that catalyze the first two reactions in the incorporation of inorganic sulfate into biomolecules. The enzymes will be purified from the filamentous fungus, Penicillium chrysogenum (which produces another important sulfur compound, penicillin.)