Evolutionary Reengineering of the Phosphofructokinase Active Site: ARG-104 Does Not Stabilize the Transition State in 6-Phosphofructo-2-Kinase
| Autor: | Yong Hwan Lee, Irwin J. Kurland, Brett Chapman, Simon J. Pilkis |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Phosphofructokinase-1
Molecular Sequence Data Biophysics Arginine Protein Engineering Polymerase Chain Reaction Biochemistry Viral Proteins Enzyme Stability medicine T7 RNA polymerase Phosphofructokinase 2 Binding site Molecular Biology Alanine Binding Sites Base Sequence biology Kinase Circular Dichroism Active site Substrate (chemistry) DNA-Directed RNA Polymerases Cell Biology Hydrogen-Ion Concentration Molecular biology Kinetics Mutagenesis Site-Directed biology.protein Phosphofructokinase medicine.drug |
| Zdroj: | Biochemical and Biophysical Research Communications. 213:663-672 |
| ISSN: | 0006-291X |
| Popis: | 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 K m for fructose-6-phosphate, no change in K m 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-P 2 ase activity without affecting substrate inhibition, which suggests that this mutation affects the bisphosphatase active site conformation and/or substrate access to it. |
| Databáze: | OpenAIRE |
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