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PubMed1993

Does the increased hydrophobicity of the interior and hydrophilicity of the exterior of an enzyme structure reflect its increased thermostability?

The values of hydrophobicity of internal and external elements of the secondary structure of three Bacillus alpha-amylase (beta alpha)8 barrel domains have been calculated in order to investigate whether there is some correlation between the values and the enzyme stability. All the values have been referred to the number of amino acids in the given beta-sheet or alpha-helix to eliminate the differences caused by non-equal length of the sheet or helix. Hydrophobicity units obtained have been averaged according to the number of internal (all beta-strands and helix alpha 7) and external (helices alpha 1-alpha 6 and alpha 8) elements of secondary structure of the alpha-amylase (beta alpha)8 barrel. The averaged hydrophobicity units have been found to correlate with the thermal stability of the three Bacillus alpha-amylases in terms of the increased hydrophobicity of the interior as well as the increased hydrophilicity of the exterior of the (beta alpha)8 barrel domain for the alpha-amylase with increased thermostability.

Bacillus↗
PubMed1987

Immobilization of glucose oxidase in poly(2-hydroxyethyl methacrylate) membranes.

Glucose oxidase (GOD) was immobilized in a poly(2-hydroxyethyl methacrylate) (HEMA) membrane through matrix entrapment in order to investigate the effect of various parameters (e.g. concentration of ingredients, temperature, repeated interaction with glucose and shelf storage) on the activity of the enzyme. Permeability of the membrane to a model permeant was tested and SEMs were obtained. It was observed that upon immobilization the affinity of GOD towards glucose was substantially decreased, and increasing the GOD content of the membrane adversely affected the activity. Membranes with the highest enzyme activity were also found to be the most permeable. Changes were detected in the pH and temperature where GOD is most active. Membrane permeability was observed to increase when crosslinker, and/or HEMA concentrations were low. The same parameters were also found to alter the morphology of the membrane as observed under SEM.

Enzyme Stability↗
PubMed1993

Glucose oxidase sandwiched between pHEMA layers: a continuous flow reactor application.

Glucose oxidase was entrapped between poly(2-hydroxyethyl methacrylate) membranes and conditions were optimized for high enzyme activity and high levels of entrapment. Highest entrapment was with a 78 microns thick coat. A continuous flow membrane reactor was designed and used. The reaction was first order with respect to glucose and to oxygen. Vmax values for the native and immobilized enzymes were 0.182 and 0.133 mM/min. The Km's for native and immobilized enzymes were 6.2 and 16.9 mM, respectively. At high substrate concentrations enzyme poisoning was detected. Both pH and temperature profiles moved to higher values upon immobilization. The enzyme retained 80% of its activity for at least 3 months in dry form.

Catalysis↗
PubMed1987

Enzyme action in polymer and salt solutions. II. Activity of penicillin acylase in poly(ethylene glycol) and potassium phosphate solutions in relation to water activity.

Increasing concentrations of poly(ethylene glycol) and potassium phosphate solutions decreased the enzymatic activity of penicillin acylase (penicillin aminohydrolase, EC 3.5.1.11). Low molecular weight poly(ethylene glycol) reduced the penicillin acylase activity more than high molecular weight poly(ethylene glycol). The decrease in enzymatic activity seemed to be related to the decrease in water activity in solutions containing poly(ethylene glycol), irrespective of the molecular weight of the polymer. The decrease in enzymatic activity in potassium phosphate solutions was lower than in poly(ethylene glycol) solutions and did not fit the water activity relationship. The decreased enzymatic activity is discussed in relation to the enzyme stability in these solutions.

Amidohydrolases↗
PubMed1987

Activity change during unfolding of bovine pancreatic ribonuclease A in guanidine.

Bovine pancreatic ribonuclease A loses almost completely its activity in 2-3 M guanidine, whereas only very slight conformational changes can be detected when following its unfolding by changes in its intrinsic fluorescence at 305 nm and ultraviolet absorbance at 287 nm. Reactivation on diluting out the denaturant is a time-dependent process, indicating that the inactivation is not due to inhibition by a reversible association of the enzyme with guanidine. The kinetic method of following the substrate reaction, in the presence of the denaturant previously proposed for use in the study of rapid inactivation reactions (Tian, W.X. and Tsou, C.-L. (1982) Biochemistry 21, 1028-1032), is applied to examine the inactivation rates of this enzyme during guanidine denaturation, and these have been compared with the unfolding rates as followed by fluorescence and absorbance changes. It is shown that during the unfolding of this enzyme in guanidine, the inactivation of the enzyme occurs within the dead time of mixing in a stopped-flow apparatus and is at least several orders of magnitude faster than the unfolding reaction as detected by the optical parameters. It appears that, as in the case of creatine kinase reported previously, the active site of a small enzyme stabilized by multiple disulfide linkages, such as ribonuclease A, is also situated in a region which is much more liable to being perturbed by denaturants than is the molecule as a whole.

Algorithms↗
PubMed1991

Purification and characterization of recombinant mouse thymidylate synthase.

Recombinant mouse thymidylate synthase (TS) expressed at high levels in Escherichia coli was purified to homogeneity in greater than 70% yield by a rapid three-step procedure. Both 0.1% Triton X-100 and 10% glycerol were required to stabilize the enzyme whose activity remained unchanged after 1 month when stored at -20 degrees C. Thermal inactivation of the enzyme was a first-order process at 37 degrees C, with t1/2 values of 6.9, 15.6 and 3.0 min at pH 5.5, 7.0 and 8.5, respectively. The presence of saturating levels of dUMP at pH 8.5 increased the t1/2 of inactivation of 38 min. The pH profile for enzyme activity showed a narrow optimum region centered at pH 7.0, which was mirrored by the shape of the Km, dUMP/Vmax plot. The pH dependence of Kd for the covalent inhibitory ternary complex of enzyme, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate exhibited a broad minimum between pH 5.5 and 8.5, and ranged between 3.1, 0.8 and 1.1 nM at pH 5.5, 7.0 and 8.5, respectively. The UV/VIS spectrum of the native enzyme exhibited a maximum at 280 nm (epsilon = 98,200 M-1 cm-1), while that of the inhibitory ternary complex showed an additional maximum at 320 nm. The 19F-NMR spectrum of the mouse enzyme:FdUMP binary complex revealed two new resonances at -2.8 and -34.8 ppm. The most deshielded resonance represented the noncovalent binary complex while the other resonance was assigned to the nucleotide covalently bound to the enzyme. The alteration of nucleotide binding equilibria produced by addition of H4 folate was exemplified by both an increase in intensity and a 5 ppm deshielding of the resonance attributed to the covalent FdUMP-enzyme complex. Addition of formaldehyde to the latter mixture produced the covalent ternary complex which resulted in the collapse of the resonances at -2.8 and -39.5 ppm and the appearance of a new resonance at -12.4 ppm.

Animals↗
PubMed1994

Analysis of progress curves for enzyme-catalyzed reactions: application to unstable enzymes, coupled reactions and transient-state kinetics.

There are several advantages to the use of progress curves to analyze the the kinetic properties of enzymes but most studies still rely on rate measurements. One of the reasons for this may be that progress curve analysis relies on the enzyme and the reactants being completely stable under assay conditions. Here a method is described that relaxes this requirement and allows progress curve analysis to be applied to unstable enzymes. The procedure is based on a combination of numerical integration and non-linear regression to fit rate equations to the progress curve data. The analysis is verified using simulated data and illustrated by application to the reaction catalyzed by alkaline phosphatase, measured in the presence of 10 mM EGTA where it has a half-life of 3 1/2 min. The method may also be applied to other experimental systems where the development over time reveals important properties but where an analytical solution of the underlying model is not known. This extension is illustrated by two systems: the coupled reactions catalyzed by pyruvate kinase and lactate dehydrogenase under conditions where both enzymes have similar activity; and the transient-state kinetics of the reaction catalyzed by glutamate dehydrogenase.

Alkaline Phosphatase↗
PubMed1996

Halolysin R4, a serine proteinase from the halophilic archaeon Haloferax mediterranei; gene cloning, expression and structural studies.

A gene encoding a halophilic serine proteinase, halolysin R4, from a halophilic archaeon Haloferax mediterranei strain R4 was cloned, its nucleotide sequence determined, and expressed in Haloferax volcanii WFD11. The deduced amino-acid sequence (403 aa in length) showed the highest similarity to halolysin 172P1, produced by another halophilic archaeon, strain 172P1 (now designated as Natrialba asiatica). Both halolysins belong to the thermitase branch of class I subtilases, but show long C-terminal extensions of 117 and 123 amino acids, respectively. Removal of this "tail' region from halolysin R4 abolished proteinase activity, indicating it provides an essential (but as yet unknown) function. Substitution of the two cysteine residues in the C-terminal extension with serine decreased enzyme stability in hypotonic solutions, possibly owing to disruption of potential disulfide bonds or perturbation of calcium binding site(s).

Amino Acid Sequence↗
PubMed1990

Protease-catalyzed synthetic reactions and immobilization-activation of the enzymes in hydrophilic organic solvents.

The catalytic feature of serine proteases for synthetic reactions in hydrophilic organic solvents and effects of immobilization by complexation with polysaccharides are described. Free alpha-chymotrypsin and subtilisin Carlsberg catalyze esterification, transesterification, and peptide synthesis in hydro-organic cosolvents with less than 10% water. Subtilisin BPN' is catalytically less active. The medium effects on the reaction kinetics and product yield were investigated in terms of the nature of solvent and water content in the reaction systems. The substrate- and stereo-specificities of the enzymes suggest that the enzymes maintain their native conformations in these low-water organic solvents. The catalytic activities of the proteases markedly increase by immobilization or complexation with polysaccharides, such as chitin or chitosan. The results of the rate measurements suggest that the primary role of the support materials is the activation of the enzymes and the increase in substrate concentration at reaction sites.

Catalysis↗
PubMed1988

Allosteric transition of aspartokinase I-homoserine dehydrogenase I studied by time-resolved fluorescence.

The allosteric transition of threonine-sensitive aspartokinase I-homoserine dehydrogenase I from Escherichia coli has been studied by time-resolved fluorescence spectroscopy. Fluorescence decay can be resolved into 2 distinct classes of tryptophan emitters: a fast component, with a lifetime of about 1.5 ns; and a slow component, with a lifetime of about 4.5 ns. The fluorescence properties of the slow component are modified by the allosteric transition. In the T-form of the enzyme stabilized by threonine, the lifetime of the slow component is longer, with a red-shifted spectrum; its accessibility to quenching by acrylamide becomes slightly higher without any decrease of fluorescence anisotropy. These results indicate a change in polarity of the slow component environment. The quaternary structure change associated with the allosteric transition probably involves global movements of structural domains without leading to any local mobility on the nanosecond time-scale. We suggest that the slow component corresponds to the unique tryptophan of the buried kinase domain.

Acrylamide↗
PubMed1986

Conductivity-mediated regulation in a compartmental enzyme system. Superactivation and conductance bistability.

This paper deals with the 'conductivity effect' which results from the action of an electric current in a compartmental system involving a chemical reaction producing ionic species. This effect, due to the difference between internal and external conductivities, leads to the accumulation of ionic species inside the reactive compartment. Different results are obtained depending on the considered kinetics: amplification of reaction rate increase, and chemical and conductance bistabilities.

Biological Transport↗
PubMed1992

Steady-state properties of a model ternary substrate cycle: theoretical predictions.

Numerous ternary substrate cycles are metabolically operative in vivo. The relative concentrations of the interconverted substrates are generally correlated with different physiological states. These cycles often include reversible and/or substrate-inhibited enzymic steps. The switch between one steady state (metabolic state) and another may be the consequence of either the effect of an exogeneous metabolite or signal, or the alteration of a cycle internal parameter. The interpretation of results obtained with currently designed experiments on substrate cycles seldom take into account the very dynamic and regulatory properties inherent in the cyclic and often autocatalytic nature of the pathway. In the present report, the various dynamic properties of a model ternary substrate cycle, bounded by moiety conservation, are investigated. Three situations with increasing complexity are considered: (i) the three enzymes are michaelian and catalyse irreversible steps; (ii) one of the enzymic steps is reversible; and (iii) one step is subjected to a destabilizing factor, i.e. inhibition by excess of substrate. The behavior(s) of the whole cycle is mainly controlled by four parameters, that is, ST, the total concentration of the substrate pool, and the three enzyme maximal velocities, VMi (i = 1,2,3). As ST (= S1 + S2 + S3) is constant, the Si steady-state concentrations (stable or not) can be represented in barycentric coordinates in a triangle (simplex). This convenient representation allows us to predict the different states of the system when one enzyme maximal activity is varied. The steady-state concentration dependencies as a function of one or several parameters may be either monostable (possibility of zero-order ultrasensitivity) or bistable (with or without reversible transitions). The physiological and experimental relevances of these observations are emphasized.

Enzyme Stability↗
PubMed1996

An analysis of the kinetics of enzymatic systems with unstable species.

We present a general kinetic analysis of the Michaelis-Menten mechanism for the case in which the substrate, the enzyme-substrate complex, and the product are unstable. The equations for the rapid equilibrium conditions are obtained as a particular case of the general equations of the transient-phase. The kinetic data analysis which we suggest is based on the time progress curve of the product of the enzymatic reaction, or on the progress curve of the species into which the immediate product is transformed. This analysis allows the determination of the rate and equilibrium constants if adequate experimental results are available. It assumes, in contrast to most previous treatments of enzyme kinetics, that the concentration of the enzyme is much higher than that of the substrate. Since this condition often is satisfied inside cells, it can be more relevant to physiological problems than the classical assumption, [E] << [S], which sooner pertains to the situation in the laboratory.

Computer Simulation↗
PubMed1991

Purification and characterization of a veratryl alcohol oxidase enzyme from the lignin degrading basidiomycete Pleurotus ostreatus.

A veratryl alcohol oxidase (VAO) enzyme was discovered in cultures of Pleurotus ostreatus. The enzyme, which oxidizes veratryl alcohol to veratraldehyde reducing O2 to H2O2, was purified to homogeneity and its main structural and catalytic properties have been determined. The enzyme is a glycoprotein and contains FAD as a prosthetic group. The amino acid composition and carboxy- and amino-terminal sequences were determined. Primary aromatic alcohols with methoxy substituents in position four are good substrates for VAO; cinnamyl alcohol is the substrate which is oxidized faster whereas coniferyl alcohol is oxidized at a slower rate. The enzyme is moderately thermostable (t1/2(55 degrees C) about 1.5 h, apparent melting temperature about 60 degrees C). The enzyme stability in 50% water/organic solvents mixtures has also been studied.

Alcohol Oxidoreductases↗
PubMed1990

Alterations in Leishmania donovani 3'-nucleotidase/nuclease activity banding pattern in polyacrylamide gels.

1. In the absence of protease inhibitors, partial purification of the 3'-nucleotidase/nuclease (3'-N'ase) from detergent extracts of Leishmania donovani leads to the appearance of bands, at 42,000 and 38,000 Da, of enzyme activity which, in SDS-PAGE, migrate faster than 43,000 Da, the apparent weight of the intact polypeptide. 2. The generation of these faster migrating species is a consequence of detergent extraction and is prevented by the addition of the protease inhibitors PMSF and leupeptin, suggesting that degradation by an endogenous protease is responsible. 3. Treatment of leishmanial membranes with exogenous proteases yields activity at 38,000 Da which is membrane-associated. Protease treatment has no effect on living cells. 4. The observed changes in enzyme migration are discussed in terms of enzyme stability and regulation. 5. The advantages and limitations of the in situ gel activity assay are considered.

Animals↗
PubMed1996

Purification and characterization of a 58,000-Da proteinase inhibitor from the hemolymph of a solitary ascidian, Halocynthia roretzi.

A new endogenous proteinase inhibitor from the cell-free hemolymph of a solitary ascidian, Halocynthia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on Ether-Toyopearl and affinity chromatography on Heparin-Sepharose. The purity of the inhibitor was examined by SDS-PAGE, gel-permeation chromatography, reversed-phase chromatography, isoelectric focusing, immunological analysis and amino-terminal amino acid sequence analysis. The inhibitor is a single polypeptide chain whose molecular weight, isoelectric point and the first 10 amino-terminal amino acid sequences are 58 kDa, pI 9.2 and NH2-Thr-Lys-Lys-Asp-Gly-Glu-Glu-Lys-Val-Ala, respectively. The purified protein inhibits plasma enzyme(s) of H. roretzi, and the rate of inhibition to the plasma enzyme(s) activity was accelerated by incubation with dextran sulfate, but the effect was neutralized by further incubation with polycation, such as polybrene or protamine sulfate. The inhibitory activity was not affected appreciably by pH 7-10 but ceased completely below pH 5 or by heating at 50 degrees C for 30 min.

Amino Acid Sequence↗
PubMed1994

Determination of phenol in urine by high-performance liquid chromatography with on-line precolumn enzymatic hydrolysis of the conjugates.

A precolumn enzyme reactor containing beta-glucosidase immobilized on LC-NH2 packed-material beads was used on-line with HPLC for determining the glucuronide/sulphate metabolites of benzene. After dilution with phosphate buffer (pH 6.8), the urine sample was injected into the HPLC system directly. Subsequently, after hydrolysis of the conjugates, phenol was produced in the enzyme reactor and was separated from other urinary components on a reversed-phase C18 column with fluorescence detection. A switching valve assembly was used to control the passage of the sample and the eluent into the reactor to prevent damage to the enzyme by the elution solvent. Factors affecting the enzymatic hydrolysis were investigated. The proposed method provides a simple and rapid procedure for urinary phenol determination. The calibration graph was linear in the range 0.25-5.0 ppm with a good correlation coefficient (r = 0.999), and in the range 0.05-1.0 ppm with r = 0.981. The detection limit was 10 ppb and the relative standard deviation was less than 2.27%. Application of the method is illustrated by the analysis of a urine sample collected from a gas station worker.

Benzene↗
PubMed1996

Aqueous two-phase systems with a liquid protein (bovine serum albumin) phase for partitioning of enzymes.

New aqueous liquid-liquid two-phase systems based on bovine serum albumin and sodium thiocyanate in combination with either poly(vinyl alcohol) or poly(ethylene glycol) were investigated. Phase diagrams are presented. Lactate dehydrogenase and some mitochondrial enzymes were partitioned in the systems. All the phase components used influenced, either positively or negatively, the activity of lactate dehydrogenase. The enzymes showed a strong preference for the albumin phase. Possible scientific and biotechnological uses are discussed.

Animals↗