Structure-Function Studies on the Sulfate Activating Enzymes

9904003西格尔，费舍尔 本研究探讨了“硫酸活化酶”ATP硫酸化酶和APS激酶的催化和调节特性的结构特征。这些酶依次催化无机硫酸盐同化过程中的前两个反应。研究了PAPS对产黄青霉同源六聚体ATP硫酸化酶的抑制作用。这种反馈过程是丝状真菌所特有的，很可能反映了PAPS是真菌中的主要分支点代谢物，而不是其他生物体的事实。真菌ATP硫酸化酶的变构域位于多肽链的C-末端区域，与APS激酶具有高度的同源性。 设计实验以确定（a）变构域的三维结构与真正APS激酶的三维结构之间的关系和（B）由变构效应物诱导的转变的结构性质。 还对同源二聚体产黄青霉APS激酶进行了研究，以建立以下结构基础：（a）对磺基核苷酸的高度特异性，（B）底物的强制有序结合（是基于动力学还是基于结构？），硫是构成活细胞的六种主要元素之一，硫的分子结构与活性中心的形成密切相关。含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）的大部分硫以无机硫酸盐形式存在。本项目的重点是ATP硫酸化酶和APS激酶的结构和功能，这两种酶催化无机硫酸盐掺入生物分子的前两个反应。这些酶将从丝状真菌产黄青霉（Penicillium chrysogenum）中纯化出来（它能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.)