guanosine-triphosphate and fructose-2-6-diphosphate

The separate bisphosphatase domain (amino acid residues 243-468) of the chicken liver bifunctional enzyme 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase was expressed in Escherichia coli and purified to homogeneity. The fructose-2, 6-bisphosphatase activity of the separate domain was 7-fold higher than that of the native bifunctional enzyme, and exhibited substrate inhibition characteristic of the native enzyme. The inhibition of the enzymes by fructose 2,6-bisphosphate could be overcome by Pi, glycerol 3-phosphate and GTP. Deletion of 30 amino acid residues from the C-terminus of the separate domain resulted in around a 5-fold increase in the Vmax of the bisphosphatase. Also, the truncated form was more accessible to chemical modification by diethyl pyrocarbonate and N-ethylmaleimide, suggesting a more open structure than the wild-type form. In addition, the mutation of cysteine-389 to alanine increased bisphosphatase activity by 20% and the Km value for fructose 2,6-bisphosphate by 3-fold, and both the point mutation at cysteine-389 and the deletional mutation led to the predominantly insoluble expression of the enzyme. The results indicated that the C-terminal tail plays a role in modulating the enzyme activity and suggested that the difference in the C-terminal tail sequence is responsible for the difference in activity of the chicken and rat liver fructose-2,6-bisphosphatases. It is postulated that an interaction between the C-terminal tail and the active site might be present.

Topics: Animals; Binding Sites; Chickens; Circular Dichroism; Diethyl Pyrocarbonate; Enzyme Inhibitors; Escherichia coli; Ethylmaleimide; Fructosediphosphates; Glycerophosphates; Guanosine Triphosphate; Kinetics; Liver; Multienzyme Complexes; Mutagenesis, Site-Directed; Peptide Fragments; Phosphofructokinase-2; Phosphoric Monoester Hydrolases; Phosphotransferases; Protein Structure, Secondary; Rats; Recombinant Proteins; Sequence Deletion; Spectrometry, Fluorescence