An Approach for Visualization of the Active Site of Enzymes with Unknown Three-Dimensional Structures

Abstract

A new approach for virtual characterization of the active site structure of enzymes with unknown three-dimensional (3D) structure has been proposed. It includes analysis of data on enzyme interaction with reversible competitive inhibitors, their 3D structures and moulding of the substrate-binding region. The superposition of ligands in biologically active conformations allows to determine the shape and dimension of the active site cavity accommodating these compounds. Monoamine oxidase A (MAO-A), a “typical” enzyme with unknown spatial organisation, was used to test this method. The correctness of such approach was validated by the analysis of HIV protease interaction with its inhibitors using 3D structures of their complexes. Mould of the substrate/inhibitor binding site can be used for the visualization of this binding site and for searching new ligands in molecular databases.     

Enzymic Formation of β-D-Fructofuranosides from Sucrose: Activity and Selectivity of Inventase in Mixtures of Water and Alcohol

Abstract

Invertase-catalysed alcoholysis of sucrose in water - primary alcohol mixtures containing up to 70% organic solvent shows 5-40% alkyl β-D-fructofuranoside formation. No formation of fructosides was observed under anhydrous conditions. The kinetics of the competing reactions of water and primary alcohol as fructosyl acceptors with sucrose as fructosyl donor were studied in order to establish the scope and limitations of the method. On a molar basis, water was a relatively unreactive acceptor. 6-Kestose formation due to sucrose itself as a fructosyl acceptor was suppressed by the presence of aliphatic alcohols. The results have been explained by assuming an nonspecific non-polar binding site to be present in the enzyme cavity near the specific polar binding site for the β-D-fructofuranosyl group of the substrate.     

Polymeric Sorbents for Bile Acids: II. Oligopeptide-Containing Resins with Quaternary Amine Groups

Abstract

Polymeric sorbents for bile acids have been prepared by attaching lysine-containing peptide sequences onto a water-swellable polyamide resin, by solid phase peptide synthesis, and then attaching a terminal N,N-dimethyl glycine residue that was subsequently quaternized. The resins with relatively longer peptide sequences demonstrated a higher binding capacity, on a per active site basis, for bile acids in pH 7.4 aqueous buffer solutions at 20°C than cholestyramine and colestipol when tested under the same in vitro conditions. In solutions of low ionic strength, they also have a degree of specificity for bile acid anions. The resins have a higher binding affinity for cholic acid than for glycocholic acid, which indicates the importance of the hydrophobic interactions in the binding.     

In silico study directed towards identification of the key structural features of GyrB inhibitors targeting MTB DNA gyrase: HQSAR,CoMSIA and molecular dynamics simulations

ABSTRACT

Mycobacterium tuberculosis DNA gyrase subunit B (GyrB) has been identified as a promising target for rational drug design against fluoroquinolone drug-resistant tuberculosis. In this study, we attempted to identify the key structural feature for highly potent GyrB inhibitors through 2D-QSAR using HQSAR, 3D-QSAR using CoMSIA and molecular dynamics (MD) simulations approaches on a series of thiazole urea core derivatives. The best HQSAR and CoMSIA models based on IC₅₀ and MIC displayed the structural basis required for good activity against both GyrB enzyme and mycobacterial cell. MD simulations and binding free energy analysis using MM-GBSA and waterswap calculations revealed that the urea core of inhibitors has the strongest interaction with Asp79 via hydrogen bond interactions. In addition, cation-pi interaction and hydrophobic interactions of the R₂ substituent with Arg82 and Arg141 help to enhance the binding affinity in the GyrB ATPase binding site. Thus, the present study provides crucial structural features and a structural concept for rational design of novel DNA gyrase inhibitors with improved biological activities against both enzyme and mycobacterial cell, and with good pharmacokinetic properties and drug safety profiles.     

Synthesis,anti-hyperglycaemic activity,and in-silico studies of N-substituted 5-(furan-2-ylmethylene)thiazolidine-2,4-dione derivatives

Thiazolidinedione derivatives have been used as anti-hyperglycemic agents in diabetic patients since last decade. In the present study, a series of N-substituted-5-(furan-2-ylmethylene)thiazolidine-2,4-dione derivatives were synthesized and characterized by ¹H-NMR, ¹³C-NMR and mass spectra. The introduction of the alkyl/haloalkyl moiety onto the amidic nitrogen of the thiazolidine-2,4-dione ring was intended to enhance the anti-hyperglycaemic activity, which was further tested in vivo by using alloxan-induced diabetic laca mice. Molecular docking simulation studies further helped in understanding the nature of the interactions and the binding mode of ligands inside the active site of the protein tyrosine phosphatase 1B enzyme, which negatively regulates the insulin signaling pathway. The compounds were screened for in-vivo anti-hyperglycaemic activity in which compounds 9 and 10 have exhibited significant decreases in blood glucose level comparable to that of pioglitazone.

     

Amperometric Enzyme-Amplified Receptor Assay For Tricyclic Antidepressants

Abstract

Enzyme-amplified receptor assay performed by competitive binding of amitriptyline drug and enzyme labeled drug for acetylcholine receptor binding sites is described. the change in enzyme activity is followed by the rate of NADH oxidation at a platinum electrode with polarizing potential of 700 mV. the response obtained using amperometric technique compares well with results from spectrophotometric studies carried out for comparison purposes.     

Study of Binding of Benzyl-Thiouracil to Human Serum Albumin by Gel Filtration Chromatography

Abstract

The binding of benzyl-thiouracil to human serum albumin was studied at 37°C and pH 7.4 by Sephadex filtration chromatography based upon Hummel and Dreyer's method. As the benzyl-thiouracil (ligand) was adsorbed on to the gel matrix, the free ligand concentrations had to be corrected through the ligand distribution between the stationary and mobile phases.

A good agreement was found between binding parameters—calculated by this method and by the classical method (equilibrium dialysis). Binding is characterized by one binding site with a moderate association constant (K₁ = 5.7 × 10⁴ M^-1) and two binding sites with a low association constant (K₂ = 7.8 × 10³ M^-1).     

Active Site Titration of Horse Liver Alcohol Dehydro-Genase by Dual Wavelength Specthophotomethy

Abstract

A simple dual wavelength spectrophotometric method for determining the active site concentration of horse liver alcohol dehydrogenase is described. The method is rapid, sensitive, accurate and requires minute amounts of enzyme. A comparison between this new application of the dual wavelength technique and the conventional methods, ordinary photometry and spectrofluorometry is furthermore discussed.     

An Evaluation of Fluorometric Substrates for Lipase

Abstract

The results of a complete study of 12 substrates for lipase indicated 4-methyl umbelliferone heptanoate to be the best substrate for the analysis of this enzyme. Using this ester, from 0.000020 to 0.066 units per ml. of porcine pancreas lipase can be determined with an accuracy and precision of about 1.5%. Analysis is performed by a direct initial reaction rate method in 2–3 minutes.     

Does Fibronect in Influence the Determination of Circulating Immune Complexes by Means of a Clq-Enzyme Immunoassay?

Abstract

Fibronect in was shown to bind to solid-phase C1q and to inhibit the binding of aggregated lgG in an C1q-solid-phase enzyme immunoassay in the absence of human serum. In the presence of human serum the determination of aggregated lgG and circulating immune complexes is not influenced by fibronectin in this assay. Therefore, we suggest that fibronectin does not affect the determination of immune complexes in serum specimens by C1q binding assays.     

Immunochemical Detection of Microquantities of Bacterial Formate Dehydrogenase

Abstract

Two immunochemical methods were developed for detection of NAD⁺?dependent formate dehydrogenase (EC 1.2.1.2, FDH) isolated from the methylotrophic bacteria Pseudomonas sp. 101:1) the dot-blot analysis using rabbit polyclonal antibodies; and 2) the indirect competitive ELISA using poly- or monoclonal mouse antibodies. The first method was used for screening the bacterial gene bank, the sensitivity is 5 and 1 pg enzyme per sample using the anti-rabbit antibodies - horse radish peroxidase conjugate or the biotinylated anti-rabbit antibodies and avidin - peroxidase conjugate, respectively. The second method was applied for precise determination of FDH concentration in cell-free extracts of selected recombinant clones. Mouse polyclonal antibodies to bacterial FDH have exibited a rather high affinity binding also to FDH from the methylotrophic yeast Candida methylica. In the indirect competitive ELISA the sensitivity of bacterial FDH determination is 1 ng per sample.     

N-Methyl Indoxyl Esters As Fluorometric Substrates For Lipase

Abstract

The results of a study of 6 N-methyl indoxyl esters as substrates for lipase indicated N-methyl indoxyl myri-state to be the best substrate for the analysis of this enzyme. Using this ester from 0.0002 to 4.0 units per ml of porcine pancreas lipase can be determined in the presence of several other esterases with an accuracy and precision of about 1.5%. Analysis is performed by a direct initial reaction rate method in 2–3 minutes.     

Fluorometric Determination of Cellulase

Abstract

A fluorometric procedure is described for the determination of the enzyme cellulase. The method is based upon the hydrolysis of the nonfluorescent substrate, resorufin acetate, by the enzyme to give the highly fluorescent resorufin (λ_ex = 540 mμ;, λ_em = 580 mμ). By this procedure from 0.00010 to 0.060 units per ml. of cellulase can be determined with an accuracy and deviation of about 1.5%. Evidence is offered to demonstrate cellulase, and not esterase, activity.     

Spectrophotofluorimetric Study of the Interaction between Trazodone Hydrochloride and Bovine Serum Albumin

Abstract

The binding of trazodone hydrochloride (TZH), an antidepressant drug, to bovine serum albumin (BSA) has been investigated by fluorescence spectroscopic analysis. The fluorescence emission of BSA (λ_em=350 nm) was quenched by TZH while that of this ligand was enhanced (λ_em=443 nm). The spectral behavior was consistent with the static quenching mechanism, and the apparent binding constant, K _a (1.05×10⁴ l mol^?1) as well as binding site number, n (～1), were estimated. Thermodynamic parameters obtained from the measured data at different temperatures showed that the binding of TZH to BSA involved predominantly hydrophobic interactions as well as smaller contributions from electrostatic forces. Phenylbutazone and ibuprofen were utilized as competitive markers for sites I and II, respectively, in the interaction of TZH with BSA. This competitive displacement procedure indicated that the likely binding was site I, i.e., subdomain IIA, and this was supported by the observation that up to 50% of this site marker, phenylbutazone, could be exchanged with TZH whilst only a few percent of ibuprofen were so affected.     

In silico identification of noncompetitive inhibitors targeting an uncharacterized allosteric site of falcipain-2

Falcipain-2 (FP-2) is a Plasmodium falciparum hemoglobinase widely targeted in the search for antimalarials. FP-2 can be allosterically modulated by various noncompetitive inhibitors that have been serendipitously identified. Moreover, the crystal structures of two inhibitors bound to an allosteric site, termed site 6, of the homolog enzyme human cathepsin K (hCatK) suggest that the equivalent region in FP-2 might play a similar role. Here, we conduct the rational identification of FP-2 inhibitors through virtual screenings (VS) of compounds into several pocket-like conformations of site 6, sampled during molecular dynamics (MD) simulations of the free enzyme. Two noncompetitive inhibitors, ZINC03225317 and ZINC72290660, were confirmed using in vitro enzymatic assays and their poses into site 6 led to calculated binding free energies matching the experimental ones. Our results provide strong evidence about the allosteric inhibition of FP-2 through binding of small molecules to site 6, thus opening the way toward the discovery of new inhibitors against this enzyme.

     

Enzyme-Amplified Receptor Assay (ERA): A Novel Approach to Drug Detection

Abstract

Isolated acetylcholine receptors from Torpedo nobiliana are used as recognition elements for phencyclidine (PCP), a potent hallucinogen with high affinity for the ion channel site associated with the receptor, in a novel enzyme-amplified receptor assay. A fixed amount of PCP labelled enzyme competes with free PCP for a limited number of receptor sites resulting in changes of observed enzyme activity proportional to free drug concentration. The proposed concept combines the use of receptors with an enzyme amplification step to yield an exceptionally simple and convenient assay.     

A Two Phase Model for the Binding of Cations To Long-Chain Polyphosphate Anions

Abstract

Binding of uni- and divalent cations to long-chain polyphosphate anions was investigated in the presence of excess uni-univalent salt. It was concluded that the binding occurs mainly in a “territorial binding” mode, i.e., most of the bound cations move freely within a specified volume around the polymer. Intrinsic complex formation (site binding) should be expressed as an equilibrium in the polymer phase.     

Thermodynamic Studies of Interaction Between Cationic Surfactants and Lysozyme Using Potentiometric Techniques

Abstract

The interaction between a homologous series of nalkyl trimethyl ammonium bromides and lysozyme was studied by a potentiometric method using a surfactant-selective electrode. The binding isotherms the interaction between a homologous series of nalkyl trimethyl ammonium bromides were measured for different conditions and show a concentration dependence attributable to aggregation of the lysozyme-surfactanl complexes. The binding isotherms arc discussed in terms of the binding potential concept of Wyman and are used to calculate an apparent Gibbs energy of binding per surfactant cation. The binding isotherms show that the C₁ decreases with increasing chain length of surfactant.     

Redox-active macrocyles designed for direct attachment to electrode surfaces

Abstract

A compound containing a redox-switchable ferrocene unit, a cation binding site (crown ether), a hydrophobic tail, and a functional group suitable for conversion into an electrode anchor has been synthesized. Its properties have been assessed by mass spectrometric and electrochemical methods and the effects of cations on the properties of this compound are presented. In addition, several hydrophobic ferrocene derivatives have been prepared and studied by the FAB/MS technique and results of those studies are also reported. These techniques demonstrate cation-mediated cooperativity between the redox center and cation binding site. Other effects resulting from the combination of subunits within a single molecule are also reported.     

Comparison Of Highly Sensitive Sandwich Enzyme Immunoassay And Radioimmunoassay For Human Ferritin

Abstract

A highly sensitive sandwich enzyme immunoassay (EIA) for human ferritin was developed using rabbit anti-ferritin IgG-coated polystyrene balls and affinity-purified rabbit anti-ferritin Fab' labelled with β-D-galactosidase from Escherichia coli and compared with the corresponding sandwich radioimmuno assay (RIA). The specific and nonspecific binding of labelled anti-ferritin to the polystyrene balls were examined in relation to the amount of labelled anti-ferritin used per tube, and the highest sensitivity of each immunoassay (0.2 amol/tube in EIA and 2.5 amol/tube in RIA) was obtained by using the minimal amount of the corresponding labelled anti-ferritin (0.71 fmol in EIA and 4.5 fmol=4436 cpm in RIA) which gave a reliable calibration curve. The sandwich RIA was less sensitive, largely because the specific radioactivity of ¹²⁵I-labelled anti-ferritin used was not sufficiently high.