Inhibition of terminal deoxynucleotidyltransferase by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate

(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate (BVdUTP), known as a specific inhibitor of herpes simplex virus (type 1)-DNA polymerase, was found to be a potent inhibitor of the activity of terminal deoxynucleotidyltransferase (TdT) from calf thymus. BVdUTP was not an efficient substrate of TdT, but it inhibited the incorporation of normal deoxynucleotide substrates in competitive fashion at the nucleotide binding site of TdT molecule. The Ki value for BVdUTP (5 microM) was much less than the Km value for dGTP (83 microM), indicating stronger affinity of the inhibitor to TdT than that of the substrate. These results indicate the usefulness of BVdUTP as a potent inhibitor of TdT for elucidation of the reaction mechanism of this enzyme.     

Incorporation of the carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate into a synthetic DNA

The carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine, C-BVDU, is a very potent and selective anti-herpes-virus compound. In order to synthesize and study the properties of a DNA that contains C-BVDU, the 5'-triphosphate, C-BVDUTP was prepared and evaluated as a potential substrate of the E. coli Klenow DNA polymerase enzyme. Although C-BVDUTP proved to be a very poor substrate also of this enzyme, it could be incorporated up to 3.6% into the synthetic DNA, poly(dA-dT, C-BVDU). This level of substitution decreased significantly the template activity for DNA and RNA polymerases, as compared to that of poly(dA-dT).     

Isolation of the glutamyl peptide labeled by the nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the active site of NADP+-specific isocitrate dehydrogenase

2-(4-Bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) is an affinity label for the coenzyme-binding site of pig heart NADP+-dependent isocitrate dehydrogenase. Specific reaction occurs at the coenzyme site with an incorporation of 0.5 mol of reagent/mol of enzyme subunit (i.e. modification of only one subunit of the dimeric enzyme) (Bailey, J.M., and Colman, R.F. (1985) Biochemistry 24, 5367-5377). Modified enzyme, prepared by incubating 1 mg/ml isocitrate dehydrogenase with 75 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of substrate or coenzyme, was reduced with NaBH4, carboxymethylated, and digested with trypsin. Nucleotidyl peptides were isolated by chromatography on DEAE-cellulose, followed by treatment with acid phosphatase (to decrease the negative charge by removing the phosphate groups from covalently bound reagent) and rechromatography on the same DEAE-cellulose column. The isolated peptides were characterized by amino acid analysis, dansylation, and gas-phase sequencing. A single triskaidekapeptide corresponding to modification of the coenzyme site by 2-BDB-T epsilon A-2',5'-DP was identified as: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Additional evidence indicated that X is a glutamate residue derivatized by 2-BDB-T epsilon A-2',5'-DP.     

Purification and characterisation of glucose (xylose) isomerase from Chainia sp. (NCL 82-5-1)

Glucose (xylose) isomerase is an important enzyme in high fructose syrup industry. The enzyme generally occurs intracellularly and is specific for both glucose and xylose. A rare actinomycete Chainia sp. (NCL 82-5-1) produces extracellular specific glucose and xylose isomerases and an intracellular glucose (xylose) isomerase. The intracellular enzyme is isolated by cell autolysis and purified by preparative polyacrylamide gel electrophoresis. Its properties are studied and compared with those of extracellular specific xylose isomerase. The intracellular enzyme has a molecular weight of 1,58,000 daltons with four equal subunits of 40,700 daltons. The N-terminal amino acid sequence analysis shows Arg at the N-terminal. Diethylpyrocarbonate inhibited the enzyme and the inhibition kinetics study shows the presence of at least 2 essential His residues. The amino acid analysis shows the absence of Cys and a high proportion of hydrophobic and acidic amino acids.     

Structure of mouse DNA (cytosine-5-)-methyltransferase

DNA (cytosine-5-)-methyltransferase was purified as a single polypeptide (190 kDa by SDS-PAGE) from mouse P815 mastocytoma cells. This enzyme transfers methyl groups to unmethylated as well as to hemimethylated DNA sites with a strong preference for the hemimethylated substrate. A structural analysis of the isolated enzyme by electron microscopical techniques was undertaken. On the basis of the results obtained, we propose a model for the enzyme structure. This model describes the enzyme as a hemi-elliptical globular structure with dimensions of 5.4-6.7 nm for the height h and 10.3-10.8 nm for the diameter d, respectively; this globular structure bears a small appendix at the flat side. A molecular mass of 235-250 kDa is calculated from the measured dimensions. Limited trypsin digestion of the enzyme led to a 160-kDa fragment which preserved the gross morphology of the original material. The possible structure function relationships are discussed.     

Iodoazomycin riboside (1-(5'-iodo-5'-deoxyribofuranosyl)-2-nitroimidazole), a hypoxic cell marker. I. Synthesis and in vitro characterization

Misonidazole (MISO), a selective radiosensitizer of hypoxic cells, forms adducts with cellular biomolecules with rates which are 30-50 X higher under hypoxic as compared to aerobic conditions of incubation. This technique of sensitizer adduct formation was proposed as a possible means of measuring the hypoxic fraction of solid tumors by noninvasive procedures. Iodoazomycin riboside (5'-IAZR) and 5'-[125I]AZR were synthesized and chemically characterized. Measurements of in vitro cytotoxicity and radiosensitizing ability with EMT-6 tumor cells in vitro indicated that 5'-IAZR is approximately 3 X more toxic and effective than is azomycin riboside (AZR) and approximately 10 X more toxic and effective than is MISO. 5'-[125I]AZR was shown to selectively bind to hypoxic EMT-6 cells at rates which were 2.5-3 X faster than those of MISO. The absolute rates of binding of 5'-IAZR to hypoxic cells at concentrations of 10-100 microM are the highest observed in this laboratory for any hypoxic cell radiosensitizer tested to date. These data suggest that 5'-IAZR, when labeled with an appropriate radioisotope (e.g., 131I), might be a useful marker for hypoxic cells in solid tumors amenable to noninvasive detection. Additional studies with animal tumor models appear to be warranted.     

1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.     

Modified polynucleotides. VI. Properties of a synthetic DNA containing the anti-herpes agent (E)-5-(2-bromovinyl)-2''-deoxyuridine

A new modified polydeoxynucleotide, a copolymer of nucleotides of 2'-deoxyadenosine and the very efficacious anti-herpesvirus agent (E)-5-(2-bromovinyl)-2'-deoxyuridine was synthesized with E. coli DNA polymerase I enzyme. It is characterized by its physical (absorption and circular dichroism spectra, thermal transition, sedimentation analysis) and bioorganic (template activity, stability) properties. Compared to poly [d(A-T)], the modified polydeoxynucleotide had a lower thermal stability but exhibited higher stability against DNases and higher template activity for DNA synthesis. Template activity for RNA synthesis of this template was, however, poor and extent of AMP and UMP incorporation was limited as well.     

5(E)-(3-azidostyryl)-2′-deoxyuridine 5′-phosphate is a photoactivated inhibitor of thymidylate synthetase

The title compound is a photoaffinity labeling reagent for thymidylate synthetase, a key enzyme for the $\text{de}\text{novo}$ biosynthesis of DNA. This compound is also a light-dependent inhibitor of murine (L-1210) and human (Namalva, Raji) tumor cell growth, and vaccinia virus replication.     

Enzymatic synthesis of mevalonate-5-(2-thiodiphosphate)

Mevalonate-5-(2-thiodiphosphate), a substrate analog for diphosphomevalonate decarboxylase, has been enzymatically prepared from mevalonate-5-phosphate and adenosine-5'-0-(3-thiotriphosphate) using phosphomevalonate kinase as a catalyst, in a 37% yield. The substrate properties of the synthesized compound are compared to those of the normal substrate of the enzyme.     

Interdependence of H+, Ca2+, and Pi (or vanadate) sites in sarcoplasmic reticulum ATPase

The phosphorylation of sarcoplasmic reticulum ATPase with Pi in the absence of Ca2+ was studied by equilibrium and kinetic experimentation. The combination of these measurements was then subjected to analysis without assumptions on the stoichiometry of the reactive sites. The analysis indicates that the species undergoing covalent interaction is the tertiary complex E X Pi X Mg formed by independent interaction of the two ligands with the enzyme. The binding constant of Pi or Mg2+ to either free or partially associated enzyme is approximately equal to 10(2) M-1, and no significant synergistic effect is produced by one ligand on the binding of the other; the equilibrium constant (Keq) for the covalent reaction E X Pi X Mg E-P X Mg is approximately equal to 16, with kphosph = 53 s-1, and khyd = 3-4 s-1 (25 degrees C, pH 6.0, no K+). The phosphorylation reaction of sarcoplasmic reticulum ATPase with Pi is highly H+ dependent. Such a pH dependence involves the affinity of enzyme for different ionization states of Pi, as well as protonation of two protein residues per enzyme unit in order to obtain optimal phosphorylation. The experimental data can then be fitted satisfactorily assuming pK values of 5.7 and 8.5 for the two residues in the nonphosphorylated enzyme (changing to 7.7 for one of the two residues, following phosphorylation) and values of 50.0 and 0.58 for the equilibrium constants of the H2(E X HPO4) in equilibrium with H(E-PO3) + H2O and H(E X HPO4) in equilibrium with E-PO3 + H2O reactions, respectively. In addition to the interdependence of H+ and phosphorylation sites, an interdependence of Ca2+ and phosphorylation sites is revealed by total inhibition of the Pi reaction when two high affinity calcium sites per enzyme unit are occupied by calcium. Conversely, occupancy of the phosphate site by vanadate (a stable transition state analogue of phosphate) inhibits high affinity calcium binding. The known binding competition between the two cations and their opposite effects on the phosphorylation reaction suggest that interdependence of phosphorylation site, H+ sites, and Ca2+ sites is a basic mechanistic feature of enzyme catalysis and cation transport.     

Purification and properties of human erythrocyte delta-amino-levulinic acid dehydratase (EC 4-2-1-24)

Human delta-aminolevulinic acid dehydratase (ALA-D) was purified 9 000-fold by salt precipitation, ion-exchange chromatography and gel filtration. These methods resulted into an electrophoretically and immunologically pure protein. The optimum pH of the enzyme is 6.6 and its Km with ALA : 4.8 X 10(-4) M. The enzymatic activity was increased by thiol-containing substances, such as dithiothreitol (DTT), which protect the -SH groups of the protein. Zinc, a portion of the enzyme molecule, was partly lost during the purification procedure; its addition enhances the enzymatic activity. Determination of molecular weights and electron microscopy study are in favor of an octameric structure.     

Thymidylate synthetase-catalyzed conversions of E-5-(2-bromovinyl)-2'-deoxyuridylate

E-5-(2-Bromovinyl)-2'-deoxyuridylate (BrvdUMP), the first metabolite in the processing of the antiviral agent E-5-(2-bromovinyl)-2'-deoxyuridine (BrvdUrd), is an excellent alternate substrate for dTMP synthetase. The nucleophilic catalyst of the enzyme adds to the 6-position of the heterocycle and converts the normally inert 5-bromovinyl group of BrvdUMP to a reactive allylic bromide in which both carbons of the side chain are susceptible to nucleophilic attack. These centers react with nucleophiles in the reaction mixture, 2-mercaptoethanol and water, to give three diastereomeric products which have been isolated and characterized. Possible implications of these findings as related to the mechanism and selectivity of BrvdUrd as an antiviral agent are discussed.     

Inhibition of thymidine kinase by P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate

Potential bisubstrate analogs, with adenosine and thymidine joined at their 5' positions by polyphosphoryl linkages of varying lengths (ApndT, where n = the number of phosphoryl groups), were examined as inhibitors of cytosolic thymidine kinase from blast cells of patients with acute myelocytic leukemia. Ki values were 1.2 microM for Ap3dT, 0.31 microM for Ap4dT, 0.12 microM for Ap5dT, and 0.19 microM for Ap6dT. The best inhibitor of the cytosolic enzyme, Ap5dT, was somewhat less effective as an inhibitor of the mitochondrial enzyme (Ki = 0.50 microM). In addition to their inhibitory modes of binding by the cytosolic enzyme, these compounds were bound at considerably lower concentrations (Kd = 0.029 microM for Ap4dT, 0.0025 microM for Ap5dT, and 0.0027 microM for Ap4dT), in such a way as to protect the cytosolic enzyme from thermal inactivation at 37 degrees C in the absence of substrates.     

N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, a novel activator of protein kinase C

Naphthalenesulfonamide derivatives were used to study the mechanism of regulation of Ca2+-dependent smooth muscle myosin light chain phosphorylation catalyzed by Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and myosin light chain kinase. Derivatives such as N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), with a hydrophobic residue at the end of a hydrocarbon chain, stimulated Ca2+-activated, phospholipid-dependent myosin light chain phosphorylation in a Ca2+-dependent fashion. There was no significant effect of these compounds on Ca2+-calmodulin (CaM) dependent myosin light chain phosphorylation. On the other hand, derivatives with the guanidino or amino residue at the same position had an inhibitory effect on both Ca2+-phospholipid- and Ca2+-CaM-dependent myosin light chain phosphorylation. These observations suggest that activation of Ca2+-activated, phospholipid-dependent myosin light chain phosphorylation by naphthalenesulfonamide derivatives depends on the chemical structure at the end of hydrocarbon chain of each compound. SC-9 was similar to phosphatidylserine with regard to activation, and the apparent Km values for Ca2+ of the enzyme with this compound and phosphatidylserine were 40 microM and 80 microM, respectively. Kinetic analysis indicated that 12-O-tetradecanoylphorbol 13-acetate increased the affinity of the enzyme with SC-9 for calcium ion. However, kinetic constants revealed that the Km value of protein kinase C activated by SC-9 for substrate myosin light chain was 5.8 microM, that is, about 10 times lower than that of the enzyme with phosphatidylserine, and that the Vmax value with SC-9 was 0.13 nmol X min-1, that is, 3-fold smaller than that seen with phosphatidylserine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity labelling of 5-aminolevulinic acid dehydratase with 2-bromo-3-(5-imidazolyl)propionic acid

2-Bromo-3-(5-imidazolyl)propionic acid, a zinc-directed thiol reagent, inactivates the enzyme 5-aminolevulinic acid dehydratase from bovine liver (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing, EC 4.2.1.24). The substrate, 5-aminolevulinic acid, completely protects against inactivation. The reagent inhibits the zinc-containing enzyme to a greater extent than the zinc-deprived enzyme; and it competes with the zinc chelator 1,10-phenanthroline. The reagent alkylates essential sulfhydryl groups of the enzyme, since the extent of the inactivation depends on the reduction of the enzyme protein by thiol compounds. It is concluded that the zinc site, the substrate site and the essential sulfhydryl groups are in close proximity in the active site.     

Purification and characterization of N-acetylglutamate 5-phosphotransferase from pea (Pisum sativum) cotyledons.

N-Acetylglutamate 5-phosphotransferase (acetylglutamate kinase, EC 2.7.2.8) has been isolated from pea (Pisum sativum) cotyledons and purified 312-fold by using heat treatment, (NH4)2SO4 fractionation, affinity chromatography on ATP--Sepharose and ion-exchange chromatography on DEAE-cellulose. This preparation was shown on polyacrylamide-gel electrophoresis to yield one band staining with Coomassie Blue. The enzyme was shown by a variety of techniques to be composed of two different kinds of subunits, of mol.wts. 43000 and 53000 respectively. These subunits are arranged to give either a dimeric or tetrameric enzyme composed of equal numbers of each type of subunit. The dimeric and tetrameric enzyme forms are thought to be interconvertible, the equilibrium between these forms being influenced by the type of ligand bound to the subunits. Kinetic studies performed on the purified enzyme, indicated a random Bi Bi type of mechanism. The enzyme displayed apparent negative co-operativity with respect to one of its substrates, N-acetylglutamate; as a result, two Km values were found for this substrate, one at 1.9 X 10(-3) M and the other at 6.2 X 10(-3) M. A single Km value for ATP was found to be 1.7 X 10(-3) M. Allosteric regulation by arginine was also shown. A model, based on the Koshland, Némethy & Filmer [(1966) Biochemistry 5, 365-385] Sequential model, which adequately describes the kinetic and structural properties of N-acetylglutamate 5-phosphotransferase, is presented.     

Characterization of cytochrome P450 2A4 and 2A5-catalyzed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen in the A/J mouse, and is believed to be a causative agent for human lung cancer. NNK requires metabolic activation by alpha-hydroxylation to exert its carcinogenic potential. The human P450, 2A6 is a catalyst of this reaction. There are two closely related enzymes in the mouse, P450 2A4 and 2A5, which differ from each other by only 11 amino acids. In the present study these two mouse P450s were expressed in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. The catalysis of NNK metabolism by Sf9 microsomal fractions containing either P450 2A4 or 2A5 was determined. Both enzymes catalyzed the alpha-hydroxylation of NNK but with strikingly different efficiencies and specificities. P450 2A5 preferentially catalyzed NNK methyl hydroxylation, while P450 2A4 preferentially catalyzed methylene hydroxylation. The KM and Vmax for the former were 1.5 microM and 4.0 nmol/min/nmol P450, respectively, and for the latter 3.9 mM and 190 nmol/min/nmol P450. The mouse coumarin 7-hydroxylase, P450 2A5 is a significantly better catalyst of NNK alpha-hydroxylation than is the closely related human enzyme, P450 2A6.     

Purification and characterization of (2S)-flavanone 3-hydroxylase from Petunia hybrida

(2S)-Flavanone 3-hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)-naringenin to (2R,3R)-dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2-oxoglutarate and Fe2+ had to be added to the buffers. The hydroxylase was purified about 200-fold by a six-step procedure with low recovery. The Mr of the enzyme was estimated by gel filtration to be about 74,000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2-oxoglutarate, Fe2+ and ascorbate. With 2-oxo[1-14C]glutarate in the enzyme assay dihydrokaempferol and 14CO2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)-(2R,3R)-dihydrokaempferol. (2S)-Naringenin, but not the (2R)-enantiomer is a substrate of the hydroxylase. (2S)-Eriodictyol is converted to (2R,3R)-dihydroquercetin. In contrast, 5,7,3',4',5'-pentahydroxy-flavanone is not a substrate. Apparent Michaelis constants for (2S)-naringenin and 2-oxoglutarate were determined to be respectively 5.6 mumol X l-1 and 20 mumol X l-1 at pH 8.5. The Km for (2S)-eriodictyol is 12 mumol X l-1 at pH 8.0. Pyridine 2,4-dicarboxylate and 2,5-dicarboxylate are strong competitive inhibitors with respect to 2-oxoglutarate with Ki values of 1.2 mumol X l-1 and 40 mumol X l-1, respectively.