Subject(s)
Antineoplastic Agents/pharmacology , Clioquinol/pharmacology , Leukemia, Experimental/drug therapy , Leukemia, Myeloid, Acute/enzymology , Multiple Myeloma/enzymology , Neoplasm Proteins/antagonists & inhibitors , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Animals , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Archaeal Proteins/antagonists & inhibitors , Asian People , Cell Line, Tumor/drug effects , Cell Line, Tumor/enzymology , Clioquinol/adverse effects , Clioquinol/therapeutic use , Copper/antagonists & inhibitors , Copper/pharmacology , Drug Screening Assays, Antitumor , Endopeptidases , Humans , Japan , K562 Cells/drug effects , K562 Cells/enzymology , K562 Cells/transplantation , Leukemia, Experimental/enzymology , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/pathology , Optic Nerve Diseases/chemically induced , Optic Nerve Diseases/ethnology , Protease Inhibitors/adverse effects , Protease Inhibitors/therapeutic use , Proteasome Endopeptidase Complex , Spinal Cord Diseases/chemically induced , Spinal Cord Diseases/ethnology , U937 Cells/drug effects , U937 Cells/enzymology , U937 Cells/transplantationABSTRACT
Golgi alpha-mannosidase II, a key enzyme in N-glycan processing, is a target in the development of anti- cancer therapies. The crystal structure of Drosophila Golgi alpha-mannosidase II in the absence and presence of the anti-cancer agent swainsonine and the inhibitor deoxymannojirimycin reveals a novel protein fold with an active site zinc intricately involved both in the substrate specificity of the enzyme and directly in the catalytic mechanism. Identification of a putative GlcNAc binding pocket in the vicinity of the active site cavity provides a model for the binding of the GlcNAcMan(5)GlcNAc(2) substrate and the consecutive hydrolysis of the alpha1,6- and alpha1,3-linked mannose residues. The enzyme-inhibitor interactions observed provide insight into the catalytic mechanism, opening the door to the design of novel inhibitors of alpha-mannosidase II.
Subject(s)
Mannosidases/chemistry , 1-Deoxynojirimycin/pharmacology , Amino Acid Sequence , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Binding Sites , Catalysis , Cell Division , Cell Line , Crystallography, X-Ray , Drosophila melanogaster/chemistry , Enzyme Inhibitors/pharmacology , Gene Expression , Mannosidases/antagonists & inhibitors , Mannosidases/genetics , Models, Molecular , Molecular Sequence Data , Neoplasm Metastasis , Protein Structure, Secondary , Substrate Specificity , Swainsonine/pharmacology , Tumor Cells, CulturedABSTRACT
We made an intracellular single-chain variable fragment (sFv) from the C219 monoclonal antibody that recognized the intracellular domain of the multidrug resistance (MDR) gene product, P-glycoprotein (P-gp). Immuno-cytochemistry using the FITC conjugated anti-C-myc tag antibody showed that the sFv protein was expressed in the cytoplasm of the cells. Although transfection of the sFv did not result in the down-regulation of P-gp expression in P-gp positive MDR cells as determined by flow cytometry analysis, Adriamycin (ADM) uptake and Rhodamine123 (Rh123) retention were increased by the C219 intra-cellular sFv transfection. The transfected cells exhibited a higher sensitivity to ADM using a 10-day colony formation assay. The conventional 3-day MTT assay showed the drug resistant tendency in C219 sFv transfected cell we tested. The growth rate of C219 sFv transfected cells was delayed in all non-MDR and MDR cells that might be the reason why C219 transfected cells exhibited the drug resistant tendency in the MTT assay. Despite this unexpected effect of C219 sFv on growth rate, our data suggest that the intra-cellular sFv technique could knockout MDR functionally and may offer a means of increasing the effectiveness of tumor chemotherapy.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/immunology , Antibodies, Monoclonal/genetics , Drug Resistance, Multiple , Immunoglobulin Fragments/genetics , Ovarian Neoplasms/immunology , ATP Binding Cassette Transporter, Subfamily B, Member 1/analysis , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Cell Division , Colony-Forming Units Assay , Cytoplasm/immunology , Doxorubicin/pharmacokinetics , Female , Flow Cytometry , Fluorescent Dyes , Gene Expression , Humans , Immunoglobulin Variable Region/genetics , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Rhodamine 123/pharmacokinetics , Transfection , Tumor Cells, CulturedABSTRACT
The ABC transporter, P-glycoprotein, is an integral membrane protein that mediates the ATP-driven efflux of drugs from multidrug-resistant cancer and HIV-infected cells. Anti-P-glycoprotein antibody C219 binds to both of the ATP-binding regions of P-glycoprotein and has been shown to inhibit its ATPase activity and drug binding capacity. C219 has been widely used in a clinical setting as a tumor marker, but recent observations of cross-reactivity with other proteins, including the c-erbB2 protein in breast cancer cells, impose potential limitations in detecting P-glycoprotein. We have determined the crystal structure at a resolution of 2.4 A of the variable fragment of C219 in complex with an epitope peptide derived from the nucleotide binding domain of P-glycoprotein. The 14-residue peptide adopts an amphipathic alpha-helical conformation, a secondary structure not previously observed in structures of antibody-peptide complexes. Together with available biochemical data, the crystal structure of the C219-peptide complex indicates the molecular basis of the cross-reactivity of C219 with non-multidrug resistance-associated proteins. Alignment of the C219 epitope with the recent crystal structure of the ATP-binding subunit of histidine permease suggests a structural basis for the inhibition of the ATP and drug binding capacity of P-glycoprotein by C219. The results provide a rationale for the development of C219 mutants with improved specificity and affinity that could be useful in antibody-based P-glycoprotein detection and therapy in multidrug resistant cancers.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/immunology , Antibodies, Monoclonal/immunology , Epitopes, B-Lymphocyte/immunology , Protein Conformation , ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Crystallography, X-Ray , Epitopes, B-Lymphocyte/chemistry , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/immunology , Ligands , Models, Molecular , Molecular Sequence DataABSTRACT
In this paper we show the organisation of the Drosophila gene encoding a Golgi alpha-mannosidase II. We demonstrate that it encodes a functional homologue of the mouse Golgi alpha-mannosidase II. The Drosophila and mouse cDNA sequences translate into amino acid sequences which show 41% identity and 61% similarity. Expression of the Drosophila GMII sequence in CHOP cells produces an enzyme which has mannosidase activity and is inhibited by swainsonine and by CuSO(4.) In cultured Drosophila cells and in Drosophila embryos, antibodies raised against a C-terminal peptide localise this product mainly to the Golgi apparatus as identified by cryo-immuno electron microscopy studies and by antibodies raised against known mammalian Golgi proteins. We discuss these results in terms of the possible use of dGMII as a Drosophila Golgi marker.
Subject(s)
Golgi Apparatus/enzymology , Mannosidases/analysis , Mannosidases/genetics , Adaptor Protein Complex alpha Subunits , Adaptor Proteins, Vesicular Transport , Animals , Antibodies , CHO Cells/physiology , CHO Cells/ultrastructure , Cloning, Molecular , Cricetinae , Drosophila , Embryo, Nonmammalian/enzymology , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation, Developmental , Genes, Insect/physiology , Golgi Apparatus/ultrastructure , Introns , Mannosidases/immunology , Membrane Proteins/analysis , Mice , Microscopy, Immunoelectron , Qa-SNARE Proteins , Swainsonine/pharmacologyABSTRACT
A construct encoding a single chain variable fragment of the anti-P-glycoprotein monoclonal antibody C219 was made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding the flexible linker (GGGGS)3, an OmpA signal sequence, a c-myc identification tag, and a five-histidine purification tag. The construct was expressed in Escherichia coli and purified from the periplasmic fraction using a nickel chelate column and ion exchange chromatography. Three-step Western blot analysis showed that the construct retains binding affinity for P-glycoprotein. Crystals of 1.0 x 0.2 x 0.2 mm were grown in 100 mM citrate, pH 4.5, 21% polyethylene glycol 6000 in the presence of low concentrations of subtilisin, resulting in proteolytic removal of the linker and purification tags. The structure was solved to a resolution of 2.4 A with an R factor of 20.6, an Rfree of 28.5, and good stereochemistry. This result could lead to a clinically useful product based on antibody C219 for the diagnosis of P-glycoprotein-mediated multidrug resistance. The molecule will also be useful in biophysical studies of functional domains of P-glycoprotein, as well as studies of the intact molecule.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/immunology , Antibodies, Monoclonal/genetics , Immunoglobulin Fragments/genetics , Immunoglobulin Variable Region/genetics , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Antibody Specificity , Cloning, Molecular , Crystallography, X-Ray , Drug Design , Escherichia coli , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/isolation & purification , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/isolation & purification , Models, Molecular , Molecular Sequence Data , Protein ConformationABSTRACT
Most glycosomal enzymes of Trypanosoma brucei carry a relatively high number of positive charges. In at least 3 of the enzymes some of the charges unique to these enzymes are concentrated in 2 distinct areas on the enzymes' surface, about 4 nm apart [4] and these positively charged structural elements have been suggested to be the site of interaction with the trypanocidal drug Suramin. We have synthesized a series of symmetrical long chain molecules with negative charges or strong dipoles at each end. Several of these compounds inhibited the glycosomal enzymes more strongly than Suramin. They also exhibited a specificity for the trypanosome enzymes, when compared with homologous enzymes from other organisms. By varying the chain length of the active compounds, a 4-nm distance between the molecules' extremes proved optimal for inhibition. Tetra-substituted compounds were better than di-substituted. Modifications introduced at the two ends indicated that a planar orientation, with an amide bond linking a phenyl ring to the chain, is preferred. Inhibition kinetics for some of the enzymes indicated the existence of multi-site interactions with the inhibitors.
Subject(s)
Benzoates/pharmacology , Dicarboxylic Acids/pharmacology , Enzyme Inhibitors/pharmacology , Glycolysis/drug effects , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/enzymology , Animals , Benzoates/chemical synthesis , Dicarboxylic Acids/chemical synthesis , Drug Design , Enzyme Inhibitors/chemical synthesis , Humans , Kinetics , Molecular Structure , Suramin/pharmacology , Trypanocidal Agents/chemical synthesis , Trypanosoma brucei brucei/drug effectsABSTRACT
Expression of the Trypanosoma brucei ornithine decarboxylase (ODC) gene in Escherichia coli behind the lambda phage PR promoter led to the production of a recombinant enzyme having the same subunit molecular weight as the native enzyme [4]. However, when the same gene is expressed behind the tac promoter or the phoA promoter, the ODCs produced by the transformed E. coli have subunit molecular weights approximately 2 kDa higher than that of the native enzyme. Amino terminal sequencing of the recombinant proteins indicates that the ODC synthesized under control of the lambda PR promoter actually starts at the second methionine (Met23) of the open reading frame, whereas those produced in the latter two cases begin at the first methionine (Met1). Analysis of the 5'-end of T. brucei ODC mRNA supports the conclusion that translation initiates at Met23. We postulate that, for the lambda PR promoter, translation initiates at Met23 instead of Met1 because of the formation of a stable secondary structure in the region of the Met1 and the presence of a good E. coli consensus translation initiation site upstream of Met23. We have constructed a new plasmid using the pho A promoter to express recombinant T. brucei ODC starting at Met23 in large quantities.
Subject(s)
Gene Expression Regulation , Ornithine Decarboxylase/genetics , Promoter Regions, Genetic , Protein Biosynthesis , Trypanosoma brucei brucei/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA Probes , DNA, Protozoan/chemistry , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Molecular Weight , Nucleic Acid Conformation , Ornithine Decarboxylase/chemistry , Plasmids , Polymerase Chain Reaction , RNA, Messenger/chemistry , RNA, Protozoan/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Trypanosoma brucei brucei/enzymologyABSTRACT
Two series of oligopeptides have been synthesized. Their effects on the activity of purified triosephosphate isomerase from Trypanosoma brucei and various other organisms have been studied. Using detailed three-dimensional structure information, the first series consisted of both cyclic and linear hydrophilic peptides that were designed to mimic the beta turns of the subunit interface loops of the trypanosome triosephosphate isomerase dimer. None of these exerted any inhibitory effect. The second series consisted of more hydrophobic cyclic peptides, originally designed to inhibit a hepatic transport system. Several of these were very effective in inhibiting the trypanosome triosephosphate isomerase, but not the homologous enzymes from rabbit, dog, yeast or Escherichia coli. The most active peptide, cyclo[-Trp-Phe-D-Pro-Phe-Phe-Lys(Z)-], exerted 50% inhibitory activity at a concentration of 3 microM. The nature of the inhibitory action of one of these compounds cyclo[-Trp-Tyr(OSO3Na)-D-Pro-Phe-Thr(OSO3Na)-Lys(Z)-] was studied in more detail. Its inhibition was noncompetitive and reversible and more than one peptide was able to bind/active site.
Subject(s)
Oligopeptides/pharmacology , Triose-Phosphate Isomerase/antagonists & inhibitors , Trypanosoma brucei brucei/enzymology , Amino Acid Sequence , Animals , Kinetics , Models, Molecular , Molecular Sequence Data , Oligopeptides/chemistry , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Solubility , Structure-Activity Relationship , Time Factors , Triose-Phosphate Isomerase/ultrastructureABSTRACT
The cloned T brucei GAPDH gene was inserted within the B subtilis GAPDH gene, carried by pUC18. Upon transformation of B subtilis by this plasmid, not able to replicate in this host, the whole plasmid was inserted in the resident chromosome, presumably by a single recombination event between homologous, chromosomal and plasmid-borne sequences. The heterologous gene was expressed, as revealed by immunological reaction with monoclonal antibodies, recognizing specifically T brucei GAPDH. T brucei GAPDH, having little or no enzyme activity, comprises about 1.56% of cellular proteins. Peptide mapping showed that a fusion of a 7.5-kDa peptide had occurred to the N-terminal part of T brucei GAPDH. This fused protein is presumably the N-terminal part of B subtilis GAPDH, in agreement with the construction of the integrative plasmid.
Subject(s)
Bacillus subtilis/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Trypanosoma brucei brucei/genetics , Animals , Antibodies, Monoclonal , Bacillus subtilis/enzymology , Blotting, Western , Cloning, Molecular , Gene Expression , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Immunoblotting , Immunoenzyme Techniques , Peptide Mapping , Plasmids , Recombinant Proteins/metabolism , Transformation, Bacterial , Trypanosoma brucei brucei/enzymologyABSTRACT
The kinetic properties of aldolase from Trypanosoma brucei were studied in comparison with aldolase from rabbit muscle and Staphylococcus aureus. The 3 enzymes displayed a similar broad pH optimum for the cleavage of fructose 1,6-bisphosphate (Fru(1,6)P2) and a similar narrow pH optimum for the cleavage of fructose 1-phosphate (Fru-1-P). However, small alterations in the maximal cleavage rate at more extreme pH values yielded disparities between the pH curves. The reaction catalyzed by the aldolases from T. brucei and S. aureus proceeded via an ordered sequence, as described for the rabbit-muscle enzyme. We determined for the 3 enzymes the kinetic parameters for both the cleavage and the formation of Fru(1,6)P2 and for the cleavage of Fru-1-P. The trypanosomal enzyme differed in its higher ratio of the maximal rate of Fru(1,6)P2-cleavage vs. the maximal rate of Fru(1,6)P2-formation, its higher affinity towards dihydroxyacetone phosphate, and its higher turnover number for the cleavage of Fru-1-P. At ionic strengths above 0.1 M the kinetic parameters of the trypanosomal enzyme followed the limited form of the Debye-Hückel equation. At ionic strengths below 0.1 M the enzyme revealed a characteristic deviation: the apparent Km for Fru(1,6)P2 increased with decreasing salt concentration. The trypanosomal aldolase was competitively inhibited by adenine nucleotides and phosphates. This inhibition occurred in the same concentration range as observed for the rabbit-muscle enzyme, while the bacterial enzyme was less affected.
Subject(s)
Bacterial Proteins/metabolism , Fructose-Bisphosphate Aldolase/metabolism , Muscles/enzymology , Staphylococcus aureus/enzymology , Trypanosoma brucei brucei/enzymology , Adenosine Diphosphate/pharmacology , Adenosine Monophosphate/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Bacterial Proteins/antagonists & inhibitors , Fructose-Bisphosphate Aldolase/antagonists & inhibitors , Fructosediphosphates/metabolism , Hydrogen-Ion Concentration , Kinetics , Muscles/drug effects , Osmolar Concentration , Phosphates/pharmacology , Rabbits , Staphylococcus aureus/drug effects , Thermodynamics , Trypanosoma brucei brucei/drug effectsABSTRACT
Pyruvate kinase from Trypanosoma brucei is a labile enzyme, losing its activity within several hours. In mixtures containing 50 mM triethanolamine buffer, pH 7.2, 25% glycerol and 0.5 mM inorganic phosphate the enzyme remained active and could be purified to homogeneity with a specific activity of 417 units mg-1 and a yield of 65%. The enzyme has an activation energy of 31.9 kJ mol-1. Magnesium and potassium ions are essential for activity. Cobalt or manganese ions replace Mg2+ but this leads to a decrease in maximal velocity. Potassium ions can be substituted by ammonium ions, while sodium ions behave as a competitive inhibitor with respect to both K+ and NH4+. All metal ions studied displayed sigmoidal kinetics. The enzyme is activated, with decreasing efficiency by fructose 2-phosphorothioate 6-phosphate, fructose 2,6-bisphosphate, fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. They all display hyperbolic kinetics. Glycerate 2,3-bisphosphate, glyceraldehyde 3-phosphate, CoASAc, oxalate, AMP, ADP, and ATP inhibit the enzyme. At substrate saturation PK was activated by Pi up to a concentration of 0.8 mM. At higher Pi concentrations the enzyme is inhibited. The enzyme is unaffected by most amino acids, only phenylalanine stimulates and tyrosine inhibits.
Subject(s)
Carbohydrate Metabolism , Pyruvate Kinase/physiology , Trypanosoma brucei brucei/enzymology , Amino Acids/pharmacology , Animals , Anions/pharmacology , Cations/pharmacology , Enzyme Stability/drug effects , Erythrocytes/drug effects , Erythrocytes/parasitology , Kinetics , Phosphates/pharmacology , Pyruvate Kinase/isolation & purification , Pyruvate Kinase/metabolism , Temperature , Trypanosoma brucei brucei/drug effects , Trypanosoma brucei brucei/growth & developmentABSTRACT
The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. Both trypanosome enzymes had pH/activity profiles similar to that of other glyceraldehyde-3-phosphate dehydrogenases, with optimal activity around pH 8.5-9. Only the yeast enzyme showed its maximal activity at a lower pH. The glycosomal enzyme was more sensitive to changes in ionic strength below 0.1 M, while the cytosolic enzyme resembled more the enzymes from rabbit muscle, human erythrocytes and yeast. The affinity for NAD of the glycosomal enzyme was 5-10-fold lower than that of the cytosolic, as well as the other enzymes. A similar, but less pronounced, difference was found for its affinity for NADH. These differences are explained by a number of amino acid substitutions in the NAD-binding domain of the glycosomal isoenzyme. In addition, the effects of suramin, gossypol, agaricic acid and pentalenolactone on the trypanosome enzymes were studied. The trypanocidal drug suramin inhibited both enzymes, but in a different manner. Inhibition of the cytosolic enzyme was competitive with NAD, while in the case of the glycosomal isoenzyme, with NAD as substrate, the drug had an effect both on Km and Vmax. The most potent inhibitor was pentalenolactone, which at micromolar concentrations inhibited the glycosomal enzyme and the enzymes from yeast and Bacillus stearothermophilus in a reversible manner, while the rabbit muscle enzyme was irreversibly inhibited.
Subject(s)
Cytosol/enzymology , Isoenzymes/metabolism , Organelles/enzymology , Trypanosoma brucei brucei/enzymology , Animals , Geobacillus stearothermophilus/enzymology , Glyceraldehyde-3-Phosphate Dehydrogenases/antagonists & inhibitors , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Humans , Hydrogen-Ion Concentration , Isoenzymes/antagonists & inhibitors , Kinetics , Muscles/enzymology , Osmolar Concentration , Rabbits , Saccharomyces cerevisiae/enzymology , Suramin/pharmacologyABSTRACT
The bacterial alkaline phosphatase (phoA) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in Escherichia coli. Other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/guanine phosphoribosyltransferase (HPRT; hypoxanthine phosphoribosyltransferase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), which carries part of the signal peptide but remains in the cytosol of the bacteria. Herein, the phoA promoter without its associated signal peptide is used to regulate expression of the HPRT of Schistosoma mansoni and the ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) of Trypanosoma brucei, two enzymes that have been identified as potential targets for antiparasitic chemotherapy. The levels of recombinant expression range from 20% to 60% of the total bacterial protein, and the majority of both recombinant enzymes was soluble. The specific activity for the recombinant trypanosomal ODC was one-third to two-thirds that of the authentic native enzyme and yields were predicted to be 15-30 mg of active enzyme per liter of bacterial culture. The specific activity for the recombinant schistosomal HPRT was equivalent to that for the native enzyme purified from schistosomes and up to 10 mg of enzymatically active HPRT has been purified from a 0.5-liter culture of treated bacteria. These results represent a break-through in recombinant expression of HPRT and ODC.
Subject(s)
Escherichia coli/genetics , Hypoxanthine Phosphoribosyltransferase/genetics , Ornithine Decarboxylase/genetics , Schistosoma mansoni/enzymology , Trypanosoma brucei brucei/enzymology , Animals , Base Sequence , Cloning, Molecular/methods , Escherichia coli/enzymology , Hypoxanthine Phosphoribosyltransferase/metabolism , Kinetics , Molecular Sequence Data , Ornithine Decarboxylase/metabolism , Plasmids , Promoter Regions, Genetic , Recombinant Proteins/metabolism , Restriction Mapping , Schistosoma mansoni/genetics , Trypanosoma brucei brucei/geneticsABSTRACT
Purified chicken liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated either from fructose 2,6-bis[2-32P]phosphate or fructose 2-phosphoro[35S]thioate 6-phosphate. The turnover of the thiophosphorylated enzyme intermediate as well as the overall phosphatase reaction was four times faster than with authentic fructose 2,6-bisphosphate. Fructose 2-phosphorothioate 6-phosphate was 10-100-fold less potent than authentic fructose 2,6-bisphosphate in stimulating 6-phosphofructo-1-kinase and pyrophosphate:fructose 6-phosphate phosphotransferase, but about 10 times more potent in inhibiting fructose 1,6-bisphosphatase. The analogue was twice as effective as authentic fructose 2,6-bisphosphate in stimulating pyruvate kinase from trypanosomes.