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

Irwin Kurland, David Geffen School of Medicine at UCLA
Brett Chapman, David Geffen School of Medicine at UCLA
Yong Hwan Lee, University of Minnesota Twin Cities
Simon Pilkis, University of Minnesota Twin Cities

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. © 1995 by Academic Press, Inc.