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Rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the kinase domain by site-directed mutagenesis.

par Crepin, K M;Vertommen, D;Dom, Geneviève ;Hue, L;Rider, M H
Référence The Journal of biological chemistry, 268, 20, page (15277-15284)
Publication Publié, 1993-07

Article révisé par les pairs

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Résumé :

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.