d-Fructose 1-Phosphate Kinase and d-Fructose 6-Phosphate Kinase from Aerobacter aerogenes

Abstract

Abstract d-Fructose 1-phosphate kinase from Aerobacter aerogenes was purified 315-fold, and its properties, regulation, and reaction kinetics were compared with those of d-fructose 6-phosphate kinase from the same organism. d-Fructose-6-P kinase exhibited a sigmoidal dependence of rate on d-fructose-6-P concentration, whereas d-fructose-1-P kinase exhibited hyperbolic dependence of rate on d-fructose-1-P concentration. The apparent Km of d-fructose-1-P kinase for either substrate (d-fructose-1-P or ATP) did not vary with the concentration of the other substrate, which indicates a sequential mechanism of substrate binding. ATP inhibited both enzymes when the ratios of Mg++ to ATP concentrations were below 2:1; ATP inhibition of d-fructose-6-P kinase was relieved by Mg++, d-fructose-6-P, ADP, and various other nucleoside diphosphates, whereas ATP inhibition of d-fructose-1-P kinase was relieved by Mg++ only. Both enzymes showed a sigmoidal dependence of rate on Mg++ concentration; increased levels of d-fructose-6-P shifted the d-fructose-6-P kinase curve from sigmoidal to hyperbolic, whereas d-fructose-1-P had no effect on a similar plot for d-fructose-1-P kinase. Other nucleoside triphosphates were used as phosphoryl donors by both enzymes and, unlike the situation with other described phosphofructokinases, inhibited activity when the ratios of Mg++ to nucleotide concentrations were below 2:1. In contrast with the result obtained with ATP, the inhibition of d-fructose-6-P kinase by other nucleoside triphosphates could not be relieved by d-fructose-6-P. The data indicate that, whereas d-fructose-6-P kinase exhibits allosteric properties and a regulatory pattern similar to those of d-fructose-6-P kinases from various other organisms, d-fructose-1-P kinase behaves more like a nonallosteric kinase. However, citrate (Ki 0.85 mm), d-fructose-1,6-di-P (Ki 4.4 mm), and d-fructose-6-P (Ki 1.0 mm) inhibited d-fructose-1-P kinase competitively with d-fructose-1-P, which suggests possible control of activity in vivo. The molecular weights of d-fructose-6-P kinase and d-fructose-1-P kinase were estimated to be 90,000 and 75,000, respectively. Other properties of the two phosphofructokinases, such as substrate specificity, Km values, pH optima, and stability, are also reported.