Site-directed mutagenesis in the effector site of Escherichia coli phosphofructokinase.
A new vector for the expression of phosphofructokinase (pfk-1) was constructed with pEMBL, which allows reliable, inducible, high-expression, and facile mutagenesis of the gene. Two mutants in the effector site of the enzyme were produced by site-specific mutagenesis of residue Tyr-55 to assess the role of its side chain in binding an allosteric inhibitor, phosphoenolpyruvate (PEP), and an activator, guanosine 5'-diphosphate (GDP): Tyr-55----Phe-55 and Try-55----Gly-55. The dissociation constant of PEP from the T state is unaffected by the mutations. Mutation of Tyr-55----Phe-55 only slightly increases the dissociation constant of GDP from the R state, indicating a minimal involvement of the hydroxyl group in binding. A 5.5-fold increase in the dissociation constant of GDP on the mutation of Tyr-55----Gly-55 suggests a small hydrophobic interaction of the aromatic ring of the tyrosine residue with guanine of GDP.
D001665:Binding Sites / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005818:Genetic Engineering; D005998:Glycine / Q000235:genetics; D006153:Guanosine Diphosphate / Q000493:pharmacokinetics / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D010728:Phosphoenolpyruvate / Q000493:pharmacokinetics / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000235:genetics*; D014443:Tyrosine / Q000235:genetics
D006153:Guanosine Diphosphate; D014443:Tyrosine; D010728:Phosphoenolpyruvate; D010732:Phosphofructokinase-1; D005998:Glycine