Molecular architecture of the glucose 1-phosphate site in ADP-glucose pyrophosphorylases
Molecular architecture of the glucose 1-phosphate site in ADP-glucose pyrophosphorylases
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DOI：
10.1074/jbc.m607088200
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发表时间：
2006-12-29
影响因子：
4.8
通讯作者：
Preiss, Jack
中科院分区：
文献类型：
--
作者：
Bejar, Clarisa Maria;Jin, Xiangshu;Preiss, Jack
ADP-Glc焦磷酸酶(PPase)是淀粉和细菌糖原生物合成途径中的关键调节酶，催化Glc-1-P和ATP合成ADP-Glc。建立了与ADP-GLC络合的大肠杆菌酶三维结构的同源模型，对底物结合部位进行了详细研究。模型中的一组氨基酸已被鉴定为与ADP-GLC配体的葡萄糖部分非常接近。通过对所设计突变体的动力学性质和热稳定性的研究，用定点突变的方法研究了这些氨基酸(Glu(194)、Ser(212)、Tyr(216)、Asp(239)、Phe(240)、Trp(274)和Asp(276))的作用。所有纯化的丙氨酸突变体对GLC-1-P的表观亲和力都比野生型低1~2个数量级，表明所选择的氨基酸在它们与底物的相互作用中起着重要作用。这些在ADP-Glc PPase家族中保守的氨基酸被其他残基取代，以研究大小、疏水性、极性、芳香性或电荷对GLC-1-P亲和力的影响。在本研究中，表征了GLC-1-P结合位点的结构。该模型与铜绿假单胞菌dTDP-Glc PPase和伤寒沙门氏菌CDP-Glc PPase等其他PPase的GLC-1-P位点重叠。因此，这里报道的数据可能会对核苷酸-二磷酸葡萄糖PPase家族的其他成员产生影响。
ADP-Glc pyrophosphorylase ( PPase), a key regulatory enzyme in the biosynthetic pathway of starch and bacterial glycogen, catalyzes the synthesis of ADP-Glc from Glc-1-P and ATP. A homology model of the three-dimensional structure of the Escherichia coli enzyme complexed with ADP- Glc has been generated to study the substrate- binding site in detail. A set of amino acids in the model has been identified to be in close proximity to the glucose moiety of the ADP- Glc ligand. The role of these amino acids (Glu(194), Ser(212), Tyr(216), Asp(239), Phe(240), Trp(274), and Asp(276)) was studied by site-directed mutagenesis through the characterization of the kinetic properties and thermal stability of the designed mutants. All purified alanine mutants had 1 or 2 orders of magnitude lower apparent affinity for Glc-1-P compared with the wild type, indicating that the selected set of amino acids plays an important role in their interaction with the substrate. These amino acids, which are conserved within the ADP- Glc PPase family, were replaced with other residues to investigate the effect of size, hydrophobicity, polarity, aromaticity, or charge on the affinity for Glc-1-P. In this study, the architecture of the Glc-1-P-binding site is characterized. The model overlaps with the Glc-1-P site of other PPases such as Pseudomonas aeruginosa dTDP-Glc PPase and Salmonella typhi CDP-Glc PPase. Therefore, the data reported here may have implications for other members of the nucleotide- diphosphoglucose PPase family.