Rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the kinase domain by site-directed mutagenesis.
Sequence alignment and modeling of the 2-kinase domain of the liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, on 6-phosphofructo-1-kinase from Bacillus stearothermophilus and Escherichia coli (Bazan, J. F., Fletterick, R. J., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646) suggested that Cys-160 of the 2-kinase would correspond to Asp-127 of the 1-kinase, which acts as a general base catalyst. We have studied the validity of this alignment by site-directed mutagenesis of residues in the 2-kinase domain of skeletal muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Cys-160 was mutated to Asp or Ser. Two adjacent residues, Glu-157 and Asp-162, either of which could act as a general base catalyst, were mutated to Ala. Asp-162 corresponds to Asp-129 in the bacterial 1-kinase, which is also essential for catalysis and might bind Mg2+. None of these mutations significantly decreased the Vmax of the 2-kinase, suggesting that the mutated amino acids are not essential for catalysis and therefore do not play the same role as Asp-127 and Asp-129 in the bacterial 1-kinase. Mutation of Glu-157 and Asp-162 to alanine had no effect on the kinetic parameters of the bifunctional enzyme, indicating that these two negatively charged residues are not involved in catalysis and substrate binding.
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Hormone and Metabolic Research Unit, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007460:Iodoacetamide / Q000494:pharmacology; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D016415:Sequence Alignment
D000596:Amino Acids; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D007460:Iodoacetamide