Separate bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: the role of the C-terminal tail in modulating enzyme activity.
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.
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Shanghai Institute of Biochemistry, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
Ling
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Wu
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Yao
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Xu
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D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D005635:Fructosediphosphates / Q000378:metabolism / Q000494:pharmacology*; D005994:Glycerophosphates / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence
D004791:Enzyme Inhibitors; D005635:Fructosediphosphates; D005994:Glycerophosphates; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C027652:fructose 2,6-diphosphate; D006160:Guanosine Triphosphate; C029620:alpha-glycerophosphoric acid; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D004047:Diethyl Pyrocarbonate; D005033:Ethylmaleimide