Dissection of the effector-binding site and complementation studies of Escherichia coli phosphofructokinase using site-directed mutagenesis.

摘要: A systematic study by site-directed mutagenesis has been conducted on the effector site of phosphofructokinase from Escherichia coli to delineate role side chains in binding allosteric activator, GDP, and inhibitor, PEP, search for key residues transtion. Target were identified crystal structure enzyme-nucleoside diphosphate complex. It is found that both activator inhibitor bind same set amino acid chains. Deletion positively charged groups (Arg21, Arg25, Arg54, Arg154, Lys213 mutated alanine) weakens effectors 2-3 kcal/mol, consistent with disruption hydrogen bonds. Residue Glu187, which known coordinated Mg2+ ion have a unique behavior mutation appears be crucial triggering transition. All other simply weaken PEP GDP parallel manner. However, Glu----Ala187 reverses roles causing become an activator. Mutation Glu----Gln187 only small effect are inhibitors. Studies described mutations different subunits tetrameric complex complement each other. The composed two subunits. In particular, Arg21 Mutations either one these abolishes activation homotetramer.(ABSTRACT TRUNCATED AT 250 WORDS)