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

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Lau, FT; Fersht, AR; Hellinga, HW; Evans, PR

Published Date

  • June 30, 1987

Published In

Volume / Issue

  • 26 / 13

Start / End Page

  • 4143 - 4148

PubMed ID

  • 2958087

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi00387a060


  • eng

Conference Location

  • United States