Mark Rider

Complete nucleotide sequence coding for rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase derived from a cDNA clone

cDNA clones for 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase were isolated from rat liver expression libraries in λgt11 by antibody, oligonucleotide, and cDNA screening. One 1860 bp long clone contained a full‐length nucleotide sequence coding for the 470 amino acids of each of the two identical subunits of the bifunctional enzyme. This clone also contained untranslated sequences, one 173 bp long upstream from the ATG start codon and one 271 bp long downstream from the TGA stop codon. The clone was terminated by a poly(A) tail of 29 nucleotides.

The ATP-binding Site in the 2-Kinase Domain of Liver 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase STUDY OF THE ROLE OF Lys-54 AND Thr-55 BY SITE-DIRECTED MUTAGENESIS

All known 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes contain a sequence (GX4GK(S/T)) in the 6-phosphofructo-2-kinase domain corresponding to the so-called nucleotide binding fold signature or Walker A motif. Mutagenesis and crystal structure data from several nucleotide binding proteins, which also contain this sequence, showed the importance of the lysine and serine/threonine residues in nucleotide binding. We have studied the role of Lys-54 and Thr-55 in MgATP binding in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis. Lys-54 was mutated to methionine, whereas Thr-55 was mutated to valine, serine, and cysteine. Three mutants, Lys-54 to Met and Thr-55 to Cys or Val, displayed more than a 5000-fold decrease in 6-phosphofructo-2-kinase activity compared with the wild type. The mutations had no effect on fructose-2,6-bisphosphatase activity and did not affect the activation of fructose-2,6-bisphosphatase after phosphorylation by cyclic 3′,5′-AMP-dependent protein kinase. Binding experiments with ATP, ADP, and their analogs (3′-N-methylanthraniloyl derivatives) showed that these two residues do not play the same role. Lys-54 is involved in ATP binding, whereas Thr-55 is important for catalysis.

Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase.

Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted.

6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase in Trypanosomatidae. Molecular characterization, database searches, modelling studies and evolutionary analysis.

Fructose 2,6‐bisphosphate is a potent allosteric activator of trypanosomatid pyruvate kinase and thus represents an important regulator of energy metabolism in these protozoan parasites. A 6‐phosphofructo‐2‐kinase, responsible for the synthesis of this regulator, was highly purified from the bloodstream form of Trypanosoma brucei and kinetically characterized. By searching trypanosomatid genome databases, four genes encoding proteins homologous to the mammalian bifunctional enzyme 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFK‐2/FBPase‐2) were found for both T. brucei and the related parasite Leishmania major and four pairs in Trypanosoma cruzi. These genes were predicted to each encode a protein in which, at most, only a single domain would be active. Two of the T. brucei proteins showed most conservation in the PFK‐2 domain, although one of them was predicted to be inactive due to substitution of residues responsible for ligating the catalytically essential divalent metal cation; the two other proteins were most conserved in the FBPase‐2 domain. The two PFK‐2‐like proteins were expressed in Escherichia coli. Indeed, the first displayed PFK‐2 activity with similar kinetic properties to that of the enzyme purified from T. brucei, whereas no activity was found for the second. Interestingly, several of the predicted trypanosomatid PFK‐2/FBPase‐2 proteins have long N‐terminal extensions. The N‐terminal domains of the two polypeptides with most similarity to mammalian PFK‐2s contain a series of tandem repeat ankyrin motifs. In other proteins such motifs are known to mediate protein–protein interactions. Phylogenetic analysis suggests that the four different PFK‐2/FBPase‐2 isoenzymes found in Trypanosoma and Leishmania evolved from a single ancestral bifunctional enzyme within the trypanosomatid lineage. A possible explanation for the evolution of multiple monofunctional enzymes and for the presence of the ankyrin‐motif repeats in the PFK‐2 isoenzymes is presented.

Inhibition of 6-phosphofructo-2-kinase activity by mercaptopurines

The activity of 6-phosphofructo-2-kinase (PFK-2), the enzyme that catalyses the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), was inhibited by mercaptopurines in vitro. Inhibition was observed with the purified enzyme from rat liver and bovine heart, and in extracts from rat lymphocytes and hepatoma cells, chick embryo fibroblasts, and human HeLa and lymphoblastoid cells. Half-maximal effect was obtained with 0.1-0.2 mM mercaptopurine and maximal inhibition ranged between 50 and 90% depending on the enzyme preparation. The inhibition resulted from a decrease in Vmax with no change in Km for ATP. The inhibition was relieved by treatment of the enzyme with thiol reducing agents, suggesting that it involves the formation of a mixed disulfide between mercaptopurine and thiol group(s) essential for enzyme activity. Incubation of intact lymphocytes or lymphoblastoid cells with 2- or 6-mercaptopurine resulted in a decrease in Fru-2,6-P2 content and lactate release. A decrease in Fru-2,6-P2 content but no change in lactate release was observed in HeLa cells and fibroblasts treated with 6-mercaptopurine but not with 2-mercaptopurine. Treatment of HeLa cells with 6-mercaptopurine resulted in a decreased PFK-2 activity which could be restored by treatment of the cell extract with dithiothreitol. In isolated rat hepatocytes and perfused rat hearts mercaptopurines had little or no effect on the Fru-2,6-P2 content and lactate release. These results suggest that the effect of 6-mercaptopurine of arresting growth in lymphoid cells might involve the inhibition of glycolysis in addition to the known inhibition of de novo purine nucleotide synthesis.

Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis.