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

Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg-138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis.

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Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Biochemical Journal ◽

10.1042/bj3230863y ◽

1997 ◽

Vol 323 (3) ◽

pp. 863-863

Author(s):

L. BERTRAND ◽

J. DEPREZ ◽

D. VERTOMMEN ◽

A. DI PIETRO ◽

L. H. HUE ◽

...

Keyword(s):

Kinase Domain ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

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Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3090341 ◽

1995 ◽

Vol 309 (1) ◽

pp. 341-346 ◽

Cited By ~ 6

Author(s):

M H Rider ◽

K M Crepin ◽

M De Cloedt ◽

L Bertrand ◽

D Vertommen ◽

...

Keyword(s):

Structural Changes ◽

Kinase Domain ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Kinetic Properties ◽

Wild Type ◽

Guanidinium Chloride ◽

Wild Type Enzyme ◽

Ph Profile ◽

Substrate Concentrations

The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2.

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Site-Directed Mutagenesis and Yeast Two-Hybrid Studies of the Insulin and Insulin-Like Growth Factor-1 Receptors: The Src Homology-2 Domain-Containing Protein hGrb10 Binds to the Autophosphorylated Tyrosine Residues in the Kinase Domain of the Insulin Receptor

Molecular Endocrinology ◽

10.1210/mend.11.12.0014 ◽

1997 ◽

Vol 11 (12) ◽

pp. 1757-1765 ◽

Cited By ~ 19

Author(s):

Lily Q. Dong ◽

Sarah Farris ◽

Jeff Christal ◽

Feng Liu

Keyword(s):

Growth Factor ◽

Kinase Domain ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Yeast Two Hybrid ◽

Tyrosine Residues ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain ◽

Two Hybrid

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Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain

Biochemical Journal ◽

10.1042/bj3000111 ◽

1994 ◽

Vol 300 (1) ◽

pp. 111-115 ◽

Cited By ~ 9

Author(s):

M H Rider ◽

K M Crepin ◽

M De Cloedt ◽

L Bertrand ◽

L Hue

Keyword(s):

Escherichia Coli ◽

Skeletal Muscle ◽

Fold Increase ◽

Kinase Domain ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Kinetic Properties ◽

Wild Type ◽

Fluorescence Measurements

Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding. The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type. Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability. The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+.

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Distinct roles of the Drosophila ninaC kinase and myosin domains revealed by systematic mutagenesis

The Journal of Cell Biology ◽

10.1083/jcb.122.3.601 ◽

1993 ◽

Vol 122 (3) ◽

pp. 601-612 ◽

Cited By ~ 81

Author(s):

JA Porter ◽

C Montell

Keyword(s):

Protein Kinase ◽

Retinal Degeneration ◽

Kinase Domain ◽

Specific Protein ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Temperature Sensitive ◽

Wild Type ◽

Multiple Functions

The Drosophila ninaC locus encodes a rhabdomere specific protein (p174) with linked protein kinase and myosin domains, required for a wild-type ERG and to prevent retinal degeneration. To investigate the role for linked kinase and myosin domains, we analyzed mutants generated by site-directed mutagenesis. Mutation of the kinase domain resulted in an ERG phenotype but no retinal degeneration. Deletion of the myosin domain caused a change in the subcellular distribution of p174 and resulted in both ERG and retinal degeneration phenotypes. Temperature-sensitive mutations in the myosin domain resulted in retinal degeneration, but no ERG phenotype. These results indicated that the ERG and retinal degeneration phenotypes were not strictly coupled suggesting that the myosin domain has multiple functions. We propose that the role of the kinase domain is to regulate other rhabdomeric proteins important in phototransduction and that the myosin domain has at least two roles: to traffic the kinase into the rhabdomeres and to maintain the rhabdomeres.

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