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.
authors.affiliation: Division of Endocrinology and Metabolism UCLA School of Medicine 90024-1736, USA.
authors.forename: I
authors.initials: I
authors.lastname: Kurland
authors.forename: B
authors.initials: B
authors.lastname: Chapman
authors.forename: Y H
authors.initials: YH
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Pilkis
mesh_terms: D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D014764:Viral Proteins
chemical_list: D014764:Viral Proteins; D001120:Arginine; D010732:Phosphofructokinase-1; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases