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

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-P₂ase activity without affecting substrate inhibition, which suggests that this mutation affects the bisphosphatase active site conformation and/or substrate access to it.