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PubMed1998

Molecular cloning and functional expression of mannitol-1-phosphatase from the apicomplexan parasite Eimeria tenella.

A metabolic pathway responsible for the biosynthesis and utilization of mannitol is present in the seven species of Eimeria that infect chickens, but is not in the avian host. Mannitol-1-phosphatase (M1Pase), a key enzyme for mannitol biosynthesis, is a highly substrate-specific phosphatase and, accordingly, represents an attractive chemotherapeutic target. Amino acid sequence of tryptic peptides obtained from biochemically purified Eimeria tenella M1Pase was used to synthesize degenerate oligonucleotide hybridization probes. Using these reagents, a partial genomic clone and full-length cDNA clones have been isolated and characterized. The deduced amino acid sequence of E. tenella M1Pase shows limited overall homology to members of the phosphohistidine family of phosphatases. This limited homology to other histidine phosphatases does, however, include several conserved residues that have been shown to be essential for their catalytic activity. Kinetic parameters of recombinant M1Pase expressed in bacteria are essentially identical to those of the biochemically purified preparation from E. tenella. Moreover, recombinant M1Pase is subject to active site-directed, hydroxylamine-reversible inhibition by the histidine-selective acylating reagent diethyl pyrocarbonate. These results indicate the presence of an essential histidine residue(s) at the M1Pase active site, as predicted for a histidine phosphatase.

Amino Acid Sequence↗

PubMed1990

Asymmetric structure of a three-arm DNA junction.

We present here experimental evidence that three-arm branched DNA molecules form an asymmetric structure in the presence of Mg2+. Electrophoretic mobility and chemical and enzymatic footprinting experiments on a three-arm branched DNA molecule formed from three 16-mer strands are described. The electrophoretic mobilities of three species of a three-arm junction in which pairs of arms are extended are found to differ in the presence of Mg2+: one combination of elongated arms migrates significantly faster than the other two. This effect is eliminated in the absence of Mg2+, leading us to suggest that the three-arm DNA junction forms an asymmetric structure due to preferential stacking of two of the arms at the junction in the presence of Mg2+. The pattern of self-protection of each 16-mer strand of the core complex exposed to Fe(II).EDTA and DNase I scission is unique, consistent with formation of an asymmetric structure in the presence of Mg2+. We conclude that three-arm junctions resemble four-arm junctions in showing preferential stacking effects at the branch site. Comparison of the scission patterns of linear duplexes and the branched trimer by the reactive probes methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2 [(OP)2CuI] further indicates that the branch point represents a site of enhanced binding for drugs, as it does in the four-arm case. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced at the branch site, consistent with loosening of base pairing or unpairing at this point.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1993

Bioluminescence of the Ca(2+)-binding photoprotein, aequorin, after histidine modification.

Modification studies of the 5 histidine residues in aequorin employing site-directed mutagenesis and diethyl pyrocarbonate suggested that His169 may be the site of binding of molecular oxygen in aequorin. The modification of this residue led to complete loss of activity, whereas modification of the remaining 4 histidine residues yielded mutant aequorins with varying bioluminescence activities.

Aequorin↗

PubMed1982

beta-Glucanases from Candida albicans: purification, characterization and the nature of their attachment to cell wall components.

beta-Glucanase activities were found associated with Candida albicans and their culture fluids. Mild acid treatment of the organisms led to rapid inactivation of beta-glucanase activities, the degree of loss increasing with the age of the cultures; the results suggested an extracytoplasmic location of the cell-associated enzymes. Most of the beta-glucanase activities were associated with the cell walls in organisms phenotypically resistant to amphotericin B methyl ester (AME). Two proteins (I and II) exhibiting beta-glucanase activity were isolated and purified by conventional procedures from cell-free extracts, cell-wall autolysates and culture fluids of C. albicans sensitive and phenotypically resistant to AME. The purified enzymes appeared homogeneous on isoelectric focusing, gel electrophoresis and ultracentrifugation, with molecular weights of 150000 (I) and 49000 (II). Both enzymes hydrolysed cell walls purified from AME-sensitive and phenotypically resistant organisms, but showed different substrate specificities and patterns of activity. Enzyme II hydrolysed (1 leads to 3)-beta-glycans by an endolytic mechanism releasing laminaritetraose as the initial product. Glucose was the only product released by enzyme I. The properties of th individual enzymes were unaffected by their localization or the age of the culture of the organisms. The loosening of the polysaccharide packing by ultrasonic treatment of cell walls purified from AME-resistant organisms increased the beta-glucanase activities bound to the walls, but did not solubilize them. Autolysis of cell walls released 58 to 66% of their beta-glucanase activity in 20 h, but no further release was attained on prolonged incubation. The amount of beta-glucanase activity released by autolysis was increased by a variety of pretreatments. Diethyl pyrocarbonate inhibited beta-glucanase activity and prevented autolysis. Evidence is presented indicating that interactions with lipids, polysaccharides and other cell wall proteins may be involved in the control of the activity of the cell wall-associated beta-glucanases in organisms phenotypically resistant to AME.

Candida albicans↗

PubMed1998

Study on splitting of sucrose glycoside bonds by beta-fructosidase of Aspergillus niger VKM F-801.

Functional groups of beta-fructosidase of Aspergillus niger VKM F-801 were identified. Based on the pH dependence of the enzyme, the calculated heat of ionization, the photoinactivation of the enzyme in the presence of methylene blue photosensitizer, and also the enzyme inactivation by diethyl pyrocarbonate, the catalytic center of the beta-fructosidase was found to include carboxyl and imidazole groups of amino acid residues. The glycoside bond in the sucrose molecule is apparently cleaved by the catalytically active carboxylate--imidazole pair.

Aspergillus niger↗

PubMed1996

Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis.

Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseoviridus P8648 to near, apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (M(r) approx. 38,000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. L-Proline-uncoupled turnover of 2-oxoglutarate to succinate and CO2 was observed. The addition of L-ascorbate did not stimulate L-proline-coupled turnover of 2-oxoglutarate, but did stimulate L-proline-uncoupled turnover. L-Ascorbate caused a time-dependent inhibition of L-proline hydroxylation. The enzyme was completely inactivated by preincubation with diethyl pyrocarbonate under histidine-modifying conditions. This inactivation could be partially prevented by the inclusion of L-proline and 2-oxoglutarate in the preincubation mixture, suggesting the presence of histidine residue(s) at the active site.

Anti-Bacterial Agents↗

PubMed1986

Inhibition of oral carcinogenesis by a protease inhibitor.

The effect of the Bowman-Birk inhibitor (BBI) and soybean trypsin inhibitor (SBTI) on experimental 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6]-induced oral carcinogenesis in Syrian male hamsters was examined. All treatments were applied topically on both cheek pouches for 20 weeks, and the animals were then sacrificed. Gross and microscopic evaluations revealed a statistically significant reduction in the number of invasive carcinomas, the total number of tumors, and the tumor mass for the DMBA + BBI treatment group compared to animals treated with DMBA alone, DMBA and autoclaved BBI (a preparation in which protease inhibitor activity is destroyed), or DMBA + SBTI. A protease activity (with the use of Boc-Val-Pro-Arg-MCA as substrate) was measured and found to be elevated about tenfold in tumorous and nontumorous tissue from DMBA-treated cheek pouches. This protease activity was found to be decreased in the DMBA and BBI treatment group but not in the DMBA + SBTI or DMBA and autoclaved BBI treatment groups, as compared to the protease activity in the DMBA treatment group. Partial characterization of the Boc-Val-Pro-Arg-MCA hydrolyzing activity with diisopropyl fluorophosphate suggests that the proteolytic activity is a serine protease. Iodoacetamide and diethyl pyrocarbonate also inhibit enzyme activity, suggesting that other residues may be necessary for catalysis, possibly including cysteine and histidine. Our results suggest that this protease activity may play a role in DMBA-induced cheek pouch carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1984

Bovine alpha- and beta-thrombin. Reduced fibrinogen-clotting activity of beta-thrombin is not a consequence of reduced affinity for fibrinogen.

beta-Thrombin, a product of the limited proteolysis of alpha-thrombin, is characterized by greatly reduced fibrinogen-clotting activity as compared to alpha-thrombin but with unchanged activity toward ester substrates. The present study was designed to elucidate the basis for the changes in the catalytic activity resulting from the conversion of bovine alpha-thrombin to bovine beta-thrombin. Fibrinogen was utilized as a competitive inhibitor in the hydrolysis of a peptide nitroanilide substrate by bovine alpha- and beta-thrombin. The Ki values obtained for fibrinogen in these experiments were similar for alpha- and beta-thrombin (about 10 microM). Similar values for Ki were obtained when fibrinogen was used to inhibit the inactivation of bovine alpha- and beta-thrombin by diisopropylphosphorofluoridate. These experiments suggested that the conversion of bovine alpha- to beta-thrombin does not affect the fibrinogen-binding site on thrombin. Differences in the reactivity of functional groups at the active site were then explored. beta-Thrombin was observed to undergo modification at the active site histidine at a slower rate than that of alpha-thrombin when reacted with either tosyllysyl chloromethyl ketone or diethyl pyrocarbonate. It is suggested that the difference in the fibrinogen-clotting activity of these two forms of thrombin can result from changes in the reactivity of the active site histidine residue.

Affinity Labels↗

PubMed1995

Spectral characterization and chemical modification of catalase-peroxidase from Streptomyces sp.

Catalase-peroxidase was purified to near homogeneity from Streptomyces sp. The enzyme was composed of two subunits with a molecular mass of 78 kDa and contained 1.05 mol of protoporphyrin IX/mol of dimeric protein. The absorption and resonance Raman spectra of the native and its cyano-enzyme were closely similar to those of other heme proteins with a histidine as the fifth ligand. However, the peak from tyrosine ring at approximately 1612 cm-1, which is unique in catalases, was not found in resonance Raman spectra of catalase-peroxidase. The electron paramagnetic resonance spectrum of the native enzyme revealed uniquely two sets of rhombic signals, which were converted to a single high spin, hexacoordinate species after the addition of sodium formate. Cyanide bound to the sixth coordination position of the heme iron, thereby converting the enzyme to a low spin, hexacoordinate species. The time-dependent inactivation of the enzyme with diethyl pyrocarbonate and its kinetic analysis strongly suggested the occurrence of histidine residue. From the above-mentioned spectroscopic results and chemical modification, it was deduced that the native enzyme is predominantly in the high spin, ferric form and has a histidine as the fifth ligand.

Catalase↗

PubMed2000

Histidine modifying agents abolish pyruvate dehydrogenase kinase activity.

Pyruvate dehydrogenase kinase (PDK) specifically phosphorylates the E1alpha subunit of the pyruvate dehydrogenase complex (PDC). Sequence analysis of cloned PDKs led to the proposal that they are mechanistically related to prokaryotic 2-component His-kinases. The reaction mechanism of protein His-kinases involves autophosphorylation of a specific His residue followed by phosphotransfer to an Asp residue. Treatment of recombinant Arabidopsis thaliana PDK with the His-directed reagents diethyl pyrocarbonate (DEPC) and dichloro-(2,2':6', 2"-terpyridine)-platinum(II) dihydrate led to a marked inhibition of autophosphorylation. In addition, DEPC treatment abolished the ability of PDK to trans-phosphorylate and inactivate PDC. These results validate the prediction that PDKs require His residues for activity.

Arabidopsis↗

PubMed1995

Function of conserved histidine residues in mammalian dihydroorotase.

Dihydroorotase (DHOase, EC 3.5.2.3) catalyzes the reversible cyclization of carbamyl aspartate to form dihydroorotate, the third step in de novo pyrimidine biosynthesis. In mammals this activity is carried by the zinc-containing domain of the 243 kDa multifunctional protein CAD. We have replaced conserved residues in the cloned 46 kDa DHOase domain by site-directed mutagenesis. Mutants His1471Ala and His1473Ala lacked catalytic activity, judging by their failure to complement a DHOase-deficient Escherichia coli strain, and were unable to coordinate the active site zinc ion in zinc blotting experiments. This result confirmed earlier predictions. A mutant protein in which the third suspected zinc ligand was changed, Glu1512Asn, had a kcat similar to that of the intact CAD molecule and a Km similar to that of the wild-type recombinant DHOase, observations that argue against a role for glutamate 1512 in catalysis. Mutant His1590Asn had no measurable catalytic activity. This histidine residue was tentatively identified as the third zinc ligand by the failure of the mutant to bind the full complement of zinc in atomic absorption measurements. Mutant His1690Asn had a kcat 34-fold lower and a Km 9-fold higher than those of wild-type recombinant. The kinetic parameters of the mutant His1642Asn were also altered, but to a lesser extent. Diethyl pyrocarbonate (DEPC) was shown previously to inactivate mammalian DHOase. Spectroscopic studies and [14C]DEPC incorporation demonstrated that the loss of activity is associated with the modification of approximately two histidine residues located at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Titration of histidine 62 in R67 dihydrofolate reductase is linked to a tetramer<-->two-dimer equilibrium.

R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded protein that confers clinical resistance to the antibacterial drug trimethoprim. To determine whether an acidic titration in kinetic pH profiles is related to titration of histidines 62, 162, 262, and 362, the stability of tetrameric R67 DHFR has been monitored as a function of pH. For the pH range 5-8, tetrameric R67 DHFR reversibly dissociates into dimers, as monitored by ultracentrifugation and molecular sieving techniques. From the crystal structures of dimeric and tetrameric R67 DHFR [Matthews et al. (1986) Biochemistry 25, 4194-4204] (Narayana, Matthews, and Xuong, personal communication), symmetry-related histidines 62, 162, 262, and 362 occur at the two dimer-dimer interfaces and protonation of these residues could destabilize tetrameric R67 DHFR. Ionization of these histidines was confirmed by monitoring the chemical shifts of the C2 proton in NMR experiments, and best fits of an incomplete titration curve yield a pKa of 6.77. Since tryptophans 38, 138, 238, and 338 also occur at the dimer-dimer interfaces, fluorescence additionally monitors the tetramer-two dimers equilibrium. When fluorescence was monitored over the pH range 5-8, a protein concentration dependence of fluorescence was observed and global fitting of three titration curves yielded Kd = 9.72 nM and pKa = 6.84 for the linked reactions: [formula: see text] Modification of H62, H162, H262, and H362 by diethyl pyrocarbonate stabilizes dimeric R67 DHFR and causes a 200-600-fold decrease in catalytic efficiency. Decreased catalytic activity in dimeric R67 DHFR is presumably due to loss of the putative single active site pore found in tetrameric R67 DHFR.

Chromatography, Gel↗

PubMed1997

Ribonuclease activity dependent cytotoxicity of Asp fl, a major allergen of A. fumigatus.

A major allergen/antigen, Asp fl, secreted by Aspergillus fumigatus exhibits cytotoxicity towards eukaryotic cell lines. Asp fl inhibited protein synthesis in RAW cells with an IC50 of 4.5 nM and also degraded ribosomal RNA of RAW cells at a similar concentration. Ribosomal inactivation by Asp fl may be the probable mechanism for protein synthesis inhibition. Specific ribonuclease activity of Asp fl was observed to be 100,000 U/mg. Presence of strong RNase activity in Asp fl was further confirmed by agar gels containing yeast RNA. Electrophoretic run on agarose gels showed that Asp fl degrades all species of naked RNA. Modification of histidine residues of Asp fl with diethyl pyrocarbonate and alkylation of cysteines with iodoacetamide resulted in loss of ribonuclease activity and cytotoxicity of Asp fl. The current study establishes the ribonuclease activity of a purified major allergen of A. fumigatus that inhibits protein synthesis and kills the eukaryotic cells.

Allergens↗

PubMed1999

Inhibition of mitochondrial permeability transition by low pH is associated with less extensive membrane protein thiol oxidation.

Ca2+ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H2O2 production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca2+ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues.

Animals↗

PubMed1997

Diadenosine polyphosphate hydrolase from presynaptic plasma membranes of Torpedo electric organ.

The diadenosine polyphosphate hydrolase present in presynaptic plasma membranes from the Torpedo electric organ has been characterized using fluorogenic substrates of the form di-(1, N6-ethenoadenosine) 5',5'''-P1,Pn-polyphosphate. The enzyme hydrolyses diadenosine polyphosphates (ApnA, where n=3-5), producing AMP and the corresponding adenosine (n-1) 5'-phosphate, Ap(n-1). The Km values of the enzyme were 0.543+/-0.015, 0.478+/-0.043 and 0. 520+/-0.026 microM, and the Vmax values were 633+/-4, 592+/-18 and 576+/-45 pmol/min per mg of protein, for the etheno derivatives of Ap3A (adenosine 5',5'''-P1,P3-triphosphate), Ap4A (adenosine 5',5"'-P1,P4-tetraphosphate) and Ap5A (adenosine 5',5'''-P1,P5-pentaphosphate) respectively. Ca2+, Mg2+ and Mn2+ are enzyme activators, with EC50 values of 0.86+/-0.11, 1.35+/-0.24 and 0.58+/-0.10 mM respectively. The fluoride ion is an inhibitor with an IC50 value of 1.38+/-0.19 mM. The ATP analogues adenosine 5'-tetraphosphate and adenosine 5'-[gamma-thio]triphosphate are potent competitive inhibitors and adenosine 5'-[alpha,beta-methylene]diphosphate is a less potent competitive inhibitor, the Ki values being 0.29+/-0.03, 0.43+/-0.05 and 7.18+/-0.8 microM respectively. The P2-receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulphonic acid behaves as a non-competitive inhibitor with a Ki value of 29.7+/-3.1 microM, and also exhibits a significant inhibitory effect on Torpedo apyrase activity. The effect of pH on the Km and Vmax values, together with inhibition by diethyl pyrocarbonate, strongly suggests the presence of functional histidine residues in Torpedo diadenosine polyphosphate hydrolase. The enzyme from Torpedo shows similarities with that of neural origin from neurochromaffin cells, and significant differences compared with that from endothelial vascular cells.

Acid Anhydride Hydrolases↗

PubMed1992

Involvement of histidine-21 in the pH-induced switch in porin channel size.

Porin is a channel-forming protein in the outer membrane of Gram-negative bacteria. In the previous paper (Todt et al., 1992), we showed that the pH induced a switch in the channel size in vitro for the porins OmpF, OmpC, and PhoE. In the results presented here, His21 of OmpC and OmpF from Escherichia coli was chemically modified with diethyl pyrocarbonate. Functional analysis of these modified porins at different pHs suggested that this histidine is involved in the pH-induced switch in channel size. Secondary structure analysis of porins at various pHs using Fourier transform infrared spectroscopy indicated that there was no global change in structure accompanying the pH-induced switch in channel size.

Bacterial Outer Membrane Proteins↗

PubMed1994

Structure and stability of human telomeric sequence.

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)n. We have investigated the structural properties of the human telomeric repeat oligonucleotide models d(T2AG3)4, d(G3T2A)3G3, and d(G3T2AG3) using CD, gel electrophoresis, and chemical probing techniques. The sequences d(G3T2A)3G3 and d(T2AG3)4 assume an antiparallel G quartet structure by intramolecular folding, while the sequence d(G3T2AG3) also adopts an antiparallel G quartet structure but by dimerization of hairpins. In all the above cases, adenines are in the loop. The TTA loops are oriented at the same end of the G tetrad stem in the case of hairpin dimer. Further, the oligonucleotide d(G3T2AG3) forms a higher order structure by the association of two hairpin dimers via stacking of G tetrad planes. Here we show that N-7 of adenine in the hairpin dimer is Hoogsteen hydrogen-bonded. The partial reactivity of loop adenines with DEPC in d(T2AG3)4 suggests that the intramolecular G quartet structure is highly polymorphic and structures with different loop orientations and topologies are formed in solution. Intra- and interloop hydrogen bonding schemes for the TTA loops are proposed to account for the observed diethyl pyrocarbonate reactivities of adenines. Sodium-induced G quartet structures differ from their potassium-induced counterparts not only in stability but also in loop conformation and interactions. Thus, the overall structure and stability of telomeric sequences are modulated by the cation present, loop sequence, and the number of G tracts, which might be important for the telomere function.

Circular Dichroism↗

PubMed1993

Guanine nucleotide-specific phosphate transfer by guanine nucleotide-binding regulatory protein beta-subunits. Characterization of the phosphorylated amino acid.

One major substrate protein was phosphorylated with [gamma-32P]GTP in membranes of human leukemia (HL-60) cells. The phosphoprotein comigrated with beta-subunits of heterotrimeric GTP-binding proteins (G proteins) in different gel systems. Upon solubilization of the phosphorylated membranes, the phosphoprotein could be immunoprecipitated by a G protein beta-subunit-specific antiserum. The beta-subunit phosphorylation was transient and was found to be specific for GTP and its analog, guanosine 5'-O-(gamma-thio)triphosphate. When phosphorylated membranes were incubated with various nucleotides, the bound phosphate was specifically removed by GDP, suggesting that the phosphate can be retransferred onto GDP. Divalent cations, preferentially Mg2+ and Mn2+, were required for both phosphorylation and dephosphorylation. The phosphorylation was stable against treatment with NaOH but sensitive to treatment with heat, HCl, and hydroxylamine. Moreover, treatment of the membranes with the histidine-modifying agent, diethyl pyrocarbonate, resulted in a loss in phosphate incorporation. The data suggest that G protein beta-subunits are involved in a guanine nucleotide-specific enzymatic activity transferring the gamma-phosphate from GTP to GDP, presumably at G protein alpha-subunits, via a phosphohistidine intermediate.

Biological Transport↗