Evolutionary Reengineering of the Phosphofructokinase Active Site: ARG-104 Does Not Stabilize the Transition State in 6-Phosphofructo-2-Kinase

Irwin Kurland, Brett Chapman, Yong Hwan Lee, Simon Pilkis

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Arg-104 of the kinase domain of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was mutated to alanine, the mutant enzyme expressed in E. coli with a T7 RNA polymerase-based expression system, and purified to homogeneity by Blue-Sepharose and Q-Sepharose chromatography. The mutant enzyme exhibited a 200-fold increase in Km for fructose-6-phosphate, no change in Km for ATP, and a 2-3-fold increase in catalytic rate. The results indicate that Arg-104, along with Arg-195, are the principal binding site residues for the 6-phosphate group of fructose-6-phosphate. In contrast to the corresponding residue in the related E. coli 6-phosphofructo-1-kinase, Arg-104 did not stabilize the transition state at pH 7-9. The Arg-104 mutation also decreased Fru-2, 6-P2ase activity without affecting substrate inhibition, which suggests that this mutation affects the bisphosphatase active site conformation and/or substrate access to it.

Original languageEnglish (US)
Pages (from-to)663-672
Number of pages10
JournalBiochemical and Biophysical Research Communications
Volume213
Issue number2
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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