Characterization of fructose 1,6-bisphosphatase from bakers' yeast.
Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000596:Amino Acids; D005635:Fructosediphosphates; D000249:Adenosine Monophosphate; D008345:Manganese; C027652:fructose 2,6-diphosphate; D006597:Fructose-Bisphosphatase; D008274:Magnesium; C029063:fructose-1,6-diphosphate