Bejar Clarisa Maria, Jin Xiangshu, Ballicora Miguel Angel, Preiss Jack
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
J Biol Chem. 2006 Dec 29;281(52):40473-84. doi: 10.1074/jbc.M607088200. Epub 2006 Nov 1.
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.
ADP - 葡萄糖焦磷酸化酶（PPase）是淀粉和细菌糖原生物合成途径中的关键调节酶，催化由Glc - 1 - P和ATP合成ADP - 葡萄糖。已构建了与ADP - 葡萄糖复合的大肠杆菌酶三维结构的同源模型，以详细研究底物结合位点。该模型中的一组氨基酸已被确定与ADP - 葡萄糖配体的葡萄糖部分紧密相邻。通过定点诱变，通过对设计突变体的动力学性质和热稳定性进行表征，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。与野生型相比，所有纯化的丙氨酸突变体对Glc - 1 - P的表观亲和力低1或2个数量级，表明所选的这组氨基酸在它们与底物的相互作用中起重要作用。这些在ADP - 葡萄糖PPase家族中保守的氨基酸被替换为其他残基，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，对Glc - 1 - P结合位点的结构进行了表征。该模型与其他PPases（如铜绿假单胞菌dTDP - 葡萄糖PPase和伤寒沙门氏菌CDP - 葡萄糖PPase）的Glc - 1 - P位点重叠。因此，本文报道的数据可能对核苷酸二磷酸葡萄糖PPase家族的其他成员有启示意义。
