Involvement of the chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444-Arg-Glu-Arg in modulation of the bisphosphatase activity by its kinase domain.
The bisphosphatase activity of the hepatic bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is repressed by its kinase domain, and regulated by cAMP-dependent protein kinase (PKA)-catalysed phosphorylation. In the present study, the mechanism by which the bisphosphatase activity is repressed by the kinase domain and regulated by phosphorylation was investigated. We found that truncation of the C-terminus of the enzyme by 25, but not 20, amino acids dramatically enhanced the catalytic rate of the bisphosphatase, abrogated the inhibition by the kinase domain, and eliminated the effect of PKA-mediated phosphorylation on activity. In addition, mutation of His444-Arg-Glu-Arg to Ala-Ala-Glu-Ala had similar effects as the deletion. Moreover, the mutations also significantly affected the phosphorylation-mediated regulation of the kinase activity of the enzyme. Furthermore, the mutations altered the pH-dependence of the bisphosphatase, and the mutant bisphosphatases were more sensitive to modification by diethyl pyrocarbonate and guanidine-induced inactivation than the wild-type enzyme. Taken together, these results demonstrate that the sequence His444-Arg-Glu-Arg plays a critical role in repression of the bisphosphatase activity by both the N-terminal kinase domain and the C-terminal tail itself. These results also explain the activation of the bisphosphatase activity by PKA-catalysed phosphorylation, by suggesting that phosphorylation may relieve the inhibitory effect of the kinase domain that is mediated by the three basic residues in this sequence.
Zhu
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Z
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Ling
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Yang
Q H
QH
Li
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004926:Escherichia coli; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D025481:Phosphofructokinase-2; D010766:Phosphorylation; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D006639:Histidine; D025481:Phosphofructokinase-2; D010732:Phosphofructokinase-1; D017868:Cyclic AMP-Dependent Protein Kinases; D006597:Fructose-Bisphosphatase; D019791:Guanidine