Kinetic studies of fructokinase I of pea seeds.
Fructokinase I of pea seeds has been purified to homogeneity and the enzyme shown to be monomeric, with a molecular weight of 72,000 +/- 4000. The reaction mechanism was investigated by means of initial velocity studies. Both substrates inhibited the enzyme; the inhibition caused by MgATP was linear-uncompetitive with respect to fructose whereas that caused by D-fructose was hyperbolic-noncompetitive against MgATP. The product D-fructose 6-phosphate caused hyperbolic-noncompetitive inhibition with respect to both substrates. MgADP caused noncompetitive inhibition, which gave intercept and slope replots that were linear with D-fructose but hyperbolic with MgATP. Free Mg2+ caused linear-uncompetitive inhibition when either substrate was varied. L-Sorbose and beta, gamma-methyleneadenosine 5'-triphosphate were used as analogs of D-fructose and MgATP, respectively. Inhibition experiments using these compounds indicated that substrate addition was steady-state ordered, with MgATP adding first. The product inhibition experiments were found to be consistent with a steady-state random release of products. The substrate inhibition caused by MgATP was most likely due to the formation of an enzyme-MgATP-product dead-end complex, whereas that caused by D-fructose was due to alternative pathways in the reaction mechanism. The inhibition caused by Mg2+ can be explained in terms of a dead-end complex with either a central complex or an enzyme-product complex.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D007887:Fabaceae / Q000201:enzymology*; D005631:Fructokinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005632:Fructose / Q000494:pharmacology; D005636:Fructosephosphates / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010770:Phosphotransferases / Q000378:metabolism*; D010946:Plants, Medicinal; D013013:Sorbose / Q000494:pharmacology; D013379:Substrate Specificity
D005636:Fructosephosphates; D005632:Fructose; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000244:Adenosine Diphosphate; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D005631:Fructokinases; D008274:Magnesium; D013013:Sorbose; C002630:alpha,beta-methyleneadenosine 5'-triphosphate