Identification of interactions that stabilize the transition state in Escherichia coli phosphofructo-1-kinase.
The activity of Escherichia coli phosphofructo-1-kinase depends upon the dissociation of a group with a pK of approximately 6.6. Mutation of the 2 active site residues most likely to be titrated in this range, Asp-127 and Asp-129, lowered activity but produced little change in the pH dependence, suggesting that these residues while important to activity were not responsible for the pH dependence of the enzyme. Alternatively, the pH dependence was thought to correspond to the negative charge on the phosphoryl group being transferred in the reaction. Mutation of Arg-72 to histidine, while lowering the activity substantially, shifted the pH optimum to approximately 6.2 with a secondary plateau on the alkaline limb that was eliminated in the double mutant R72H/R171H. Mutation of Arg-171 alone produced only a small decrease in maximal activity and little change in pH dependence from the wild type enzyme. The data support an associative mechanism with a transition state stabilized by the interaction between the negative charge on the phosphoryl group being transferred and the arginyl residue at position 72 on the enzyme.
Zheng
R L
RL
Department of Biological Chemistry, Chicago Medical School, North Chicago, Illinois 60064.
Kemp
R G
RG
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase