Role of the C-terminal region in the allosteric properties of Escherichia coli phosphofructokinase-1.
In order to investigate the role of the carboxy-terminal segment in the catalytic, regulatory, and structural properties of the major allosteric phosphofructokinase (ATP:D-fructose-6-phosphate-1-phosphotransferase: EC 2.7.1.11) from Escherichia coli, the corresponding gene has been modified at either of two sites using oligonucleotide-directed mutagenesis: the codon at position 279 was changed from TAC (Tyr) into TAA (Ochre), and the codon at position 311 from TGG (Trp) into TAG (Amber). The gene mutated at position 279 is not expressed as an active enzyme, probably because a polypeptide chain lacking 41 C-terminal residues cannot fold and/or assemble under the intracellular conditions. The gene mutated at position 311 is expressed as an active enzyme which has been purified to homogeneity. The fluorescence of this protein shows that it has no tryptophan, which confirms that the last nine residues at the carboxy terminal are missing. This derivative has almost the same specific activity and affinities for the two substrates (fructose-6-phosphate and ATP) as intact phosphofructokinase; the saturation by fructose 6-phosphate is also very cooperative. The last nine residues are thus not important for substrate binding, homotropic cooperativity, and catalytic efficiency. The activity of the mutant enzyme is still sensitive to activation by GDP or inhibition by phosphoenolpyruvate, but its affinity for the allosteric effectors is reduced. The carboxy-terminal segment also appears to contribute to the stability of the interactions between subunits: the mutant protein is less stable than the wild type towards denaturation by heat or guanidinium hydrochloride.
Serre
M C
MC
Laboratoire d'Enzymologie, C.N.R.S., Gif-sur-Yvette, France.
Garel
J R
JR
D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005809:Genes, Regulator; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D011487:Protein Conformation; D015202:Protein Engineering; D013050:Spectrometry, Fluorescence
D010732:Phosphofructokinase-1