Purification and properties of phosphofructokinase (EC 2.7.1.11) of Saccharomyces carlsbergensis.
A procedure for the purification of phosphofructokinase from brewer's yeast (Saccharomyces carlsbergensis) is reported. Treatments with organic solvents and heat were avoided and chromatographic and filtration techniques in the presence of phenylmethane sulfonyl fluoride were mainly used. The purified enzyme is homogeneous in disc gel electrophoresis and according to sedimentation velocity and equilibrium measurements in the ultracentrifuge. The isoelectric point determined by focusing was 5.3. Absorption spectra, fluorescence spectra and circular dichroism spectrum are given. The molecular weight of the purified enzyme determined by gel filtration was 720 000, in agreement with that of the enzyme in the raw extract. This confirms the results of sedimentation velocity experiments which gave a value of SO20, W equals 19.4. Alkaline treatment leads to a dissociation of the native enzyme, yielding an inactive species with a molecular weight of 360 000. In 6M guanidine hydrochloride the enzyme dissociates into subunits with a mean molecular weight of 90 000 as obtained by ultracentrifugation analysis. This suggests a structure composed of 8 monomers. The specific activity of the enzyme was 116 U/mg under optimum conditions. The enzyme activity was proportional to the enzyme concentration in the range of 6 times 10- minus 12 M to 3 times 10- minus 7 M. The Michaelis constants and Hill coefficients for fructose 6-phosphate and AMP, the pH optima, and the stability properties of the enzyme are reported. Furthermore, the activation energy is given and it is shown that under saturating conditions, a straight Arrhenius plot obtains, whereas the plot is discontinuous at high ATP concentrations and at pH 7.6.
D000227:Adenine Nucleotides; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004589:Electrophoresis, Disc; D005453:Fluorescence; D005636:Fructosephosphates; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000302:isolation & purification*; D011489:Protein Denaturation; D012440:Saccharomyces / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014461:Ultracentrifugation
D000227:Adenine Nucleotides; D005636:Fructosephosphates; D010732:Phosphofructokinase-1