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

Irwin J. Kurland, B. Chapman, Y. H. Lee, S. Pilkis

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6 Citations (Scopus)

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 - Aug 15 1995
Externally publishedYes

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Phosphofructokinase-2
Phosphofructokinases
Reengineering
Escherichia coli
Catalytic Domain
Phosphofructokinase-1
Agarose Chromatography
Mutation
Substrates
Enzymes
Chromatography
Alanine
Sepharose
Conformations
Phosphotransferases
Adenosine Triphosphate
Phosphates
Binding Sites
fructose-6-phosphate

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology
  • Biophysics
  • Biochemistry

Cite this

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title = "Evolutionary Reengineering of the Phosphofructokinase Active Site: ARG-104 Does Not Stabilize the Transition State in 6-Phosphofructo-2-Kinase",
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.",
author = "Kurland, {Irwin J.} and B. Chapman and Lee, {Y. H.} and S. Pilkis",
year = "1995",
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T2 - ARG-104 Does Not Stabilize the Transition State in 6-Phosphofructo-2-Kinase

AU - Kurland, Irwin J.

AU - Chapman, B.

AU - Lee, Y. H.

AU - Pilkis, S.

PY - 1995/8/15

Y1 - 1995/8/15

N2 - 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.

AB - 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.

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