Site-directed mutagenesis in the effector site of Escherichia coli phosphofructokinase

Biochemistry. 1987 Jun 30;26(13):4143-8. doi: 10.1021/bi00387a060.

Abstract

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.

Publication types

  • Comparative Study

MeSH terms

  • Binding Sites* / drug effects
  • Enzyme Activation / drug effects
  • Escherichia coli / enzymology*
  • Genetic Engineering
  • Glycine / genetics
  • Guanosine Diphosphate / pharmacokinetics
  • Guanosine Diphosphate / pharmacology
  • Kinetics
  • Mutation
  • Phosphoenolpyruvate / pharmacokinetics
  • Phosphoenolpyruvate / pharmacology
  • Phosphofructokinase-1 / antagonists & inhibitors
  • Phosphofructokinase-1 / genetics*
  • Tyrosine / genetics

Substances

  • Guanosine Diphosphate
  • Tyrosine
  • Phosphoenolpyruvate
  • Phosphofructokinase-1
  • Glycine