Fructose 1,6-Diphosphatase from Rabbit Liver. VI. Functional Tyrosyl Residues in the Active Center * (original) (raw)
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Biochemistry, 1966
Treatment of crystalline rabbit liver fructose diphosphatase (FDPase) with N-acetylimidazole results in a time-dependent 0-acetylation of ten tyrosyl residues. In the first phase of the reaction two to three tyrosyl residues are acetylated with no change in catalytic properties. The acetylation of four additional residues is associated with loss of allosteric inhibition by adenosGe monophosphate (AMP). Finally, with
Univalent cation activation of fructose 1,6-diphosphatase
Archives of Biochemistry and Biophysics, 1970
Fructose 1,6-diphosphatase from several vertebrate sources has been studied with respect to univalent cation activation. All the enzymes tested showed activation by certain univalent cations, K+ or NH,+ being the best activators. A re-evaluation of some properties of fructose 1,6-diphosphatases in the presence of univalent cation activators showed, as studied with pig kidney, rabbit liver, and rabbit muscle fructose 1,6-diphosphatase, that the MgZ+-saturation curves were markedly altered by the presence of 150 mu K+. Not only an increase in K, and Tim,, was observed, but also the sigmoidal nature of the Mg?saturation curves became evident. AMP inhibition, characteristic of most fructose 1,6-diphosphatases, was not significantly altered by the presence of K+ in the case of pig kidney, rabbit liver, and fish (Roja chilensis) liver fructose 1,6-diphosphatase. Rabbit muscle fructose 1,6-diphosphatase became more sensitive to AMP inhibition, while in the case of fish (Genipterus chilensis) liver fructose 1,6-diphosphatase, inhibition by AMP could only be demonstrated in the presence of the univalent cation activators.
Journal of protein …, 1993
262, 8451-8454]. On the basis of these results, it was suggested that a single reactive sulfhydryl group was essential for the inhibition. We have isolated a peptide bearing the N-ethylmaleimide target site and the modified residue has been identified as cysteine-128. We have further examined the reactivity of this group and demonstrated that when reagents with bulky groups are used to modify the protein at the reactive sulfhydryl [e.g., N-ethylmaleimide or 5,5'dithiobis-(2-nitrobenzoate)], most of the fructose 2,6-bisphosphate inhibition potential is lost. However, there is only partial or no loss of inhibition when smaller groups (e.g., cyanate or cyanide) are introduced. Kinetic and ultraviolet difference spectroscopy-binding studies show that the treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide causes a considerable reduction in the affinity of the enzyme for fructose 2,6-bisphosphate while affinity for fructose 1,6-bisphosphate does not change. We can conclude that modification of this reactive sulfhydryl affects the enzyme sensitivity to fructose 2,6-bisphosphate inhibition by sterically interfering with the binding of this sugar bisphosphate, although this residue does not seem to be essential for the inhibition to occur. The results also suggest that fructose 1,6-bisphosphate and fructose 2,6-bisphosphate may interact with the enzyme in a different way.
Fructose-2,6-bisphosphatase from rat liver
European journal of biochemistry / FEBS, 1982
An enzyme that catalyzes the stoichiometric conversion of fructose 2,6-bisphosphate into fructose 6-phosphate and inorganic phosphate has been purified from rat liver. This fructose 2,6-bisphosphatase copurified with phosphofructokinase 2 (ATP: D-fructose 6-phosphate 2-phosphotransferase) in the several separation procedures used. The enzyme was active in the absence of Mg2+ and was stimulated by triphosphonucleotides in the presence of Mg2+ and also by glycerol 3-phosphate, glycerol 2-phosphate and dihydroxyacetone phosphate. It was strongly inhibited by fructose 6-phosphate at physiological concentrations and this inhibition was partially relieved by glycerol phosphate and dihydroxyacetone phosphate. The activity of fructose 2,6-bisphosphatase was increased severalfold upon incubation in the presence of cyclic-AMP-dependent protein kinase and cyclic AMP. The activation resulted from an increase in V (rate at infinite concentration of substrate) and from a greater sensitivity to th...