Abstract
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+.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Aspartic Acid / genetics
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Aspartic Acid / metabolism*
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Base Sequence
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Blotting, Western
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Chromatography, Liquid
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Electrophoresis, Polyacrylamide Gel
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Fluorescence Polarization
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Kinetics
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Molecular Sequence Data
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Muscles / enzymology*
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Mutagenesis, Site-Directed
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Oligodeoxyribonucleotides
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Phosphofructokinase-2
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Phosphoric Monoester Hydrolases / genetics
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Phosphoric Monoester Hydrolases / isolation & purification
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Phosphoric Monoester Hydrolases / metabolism*
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Phosphotransferases (Alcohol Group Acceptor) / genetics
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Phosphotransferases (Alcohol Group Acceptor) / isolation & purification
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Phosphotransferases (Alcohol Group Acceptor) / metabolism*
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Protein Conformation
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Rats
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Sequence Alignment
Substances
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Oligodeoxyribonucleotides
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Aspartic Acid
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Phosphotransferases (Alcohol Group Acceptor)
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Phosphofructokinase-2
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Phosphoric Monoester Hydrolases