Purification and properties of 1-phosphofructokinase from Escherichia coli

1-Phosphofructokinase has been purified from Escherichia coli. Size exclusion chromatography revealed an Mr of 57000. After sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) we found a single protein band corresponding to an Mr of 30000. The enzyme was highly specific for fructose-1-phosphate. ATP and GTP were the best phosphoryl donors, whereas UTP and CTP showed only a weak effect. The enzyme was reversibly inhibited by fructose-1, 6-bisphosphate and ADP and irreversibly by phenyl-methanesulfonyl fluoride (PMSF). ATP prevented irreversible inhibition by PMSF. Fructose-1-phosphate had a much less pronounced effect on irreversible inhibition by PMSF. Comparison of the peptide map of tryptic digested 1-phosphofructokinase and PMSF-modified 1-phosphofructokinase revealed an additional peak for the modified 1-phosphofructokinase whereas the intensity of another peak was lowered compared to untreated 1-phosphofructokinase. This peptide might therefore represent the binding site for ATP.