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A Schellenberger

Publications and source records attributed to A Schellenberger.

At least 37 records · Page 2Linked to original sources
PubMed1988

The functional role of thiol groups of pyruvate decarboxylase from brewer's yeast.

Pyruvate decarboxylase purified from brewer's yeast has been modified by the thiol specific reagents 4-hydroxy-mercuri-benzoate and 3-bromo-pyruvamide. The kinetic properties of the thiol-modified enzyme derivatives were investigated by stopped-flow technique. The enzyme--inactive in the absence of its substrate--is activated by binding pyruvate to the regulatory sites. This activation behaviour is lost after modification of six thiol groups per PDC molecule. The thiol groups have been subdivided into two classes according to their modification rates. Two of these six thiol groups are involved in the mechanism of enzyme activation.

Carboxy-Lyases↗
PubMed1987

Effects of ultrasound on the catalytic activity of matrix-bound glucoamylase.

The effect of ultrasonic waves on the activity of glucoamylase bound to a porous polystyrene matrix is investigated in this Paper. The immobilized enzyme was sonated in a flow cuvette at frequencies between 1 and 11 MHz and sound intensities up to 5 kW m-2. The effect was measured as a function of the type and concentration of the substrate, carrier particle size, flow rate of the substrate solution and ultrasonic frequency. The activity increase is discussed in terms of a possible ultrasonic mechanism.

Catalysis↗
PubMed1986

Investigations on the pyruvate decarboxylase catalysed oxidative decarboxylation of 2-oxoacids by 2.6-dichlorophenolindophenol.

Pyruvate decarboxylase, a thiamine pyrophosphate and Mg2+ dependent enzyme, catalyzes normally the simple decarboxylation of its substrate. However, in the presence of suitable hydrogen acceptors, such as dichlorophenolindophenol, it catalyzes an artificial reaction, the oxidative decarboxylation of 2-oxoacids to the corresponding carboxylic acids. As a result of kinetic studies a mechanism is presented in this paper, which describes the synchronous progress of both the oxidative ping-pong-type decarboxylation reaction and the physiological non-oxidative decarboxylation reaction. Moreover, experiments using phenylglyoxylic acids that carry suitable substituents in the 4-position have shown that the electronic influence of the substituents (causing a decrease in the rate constants with increasing electron pressure) is in quantitative agreement for both types of reactions. A common rate limiting transition state preceding the alpha-carbanion intermediate of the enzymic reaction (2-(1-hydroxybenzyl)-thiamine pyrophosphate carbanion) must therefore be assumed for both reactions. Acetaldehyde which acts as noncompetitive inhibitor in the normal enzyme mechanism does not influence the oxidative decarboxylation reaction. 4'-hydroxy-4'-deamino-thiamine pyrophosphate is inactive as coenzyme for both types of enzyme reactions. This confirms again the essential role of the 4'-amino group in the cofactor function.

2,6-Dichloroindophenol↗
PubMed1985

The effect of thiamine pyrophosphate modification on its coenzyme function in a transketolase-catalyzed reaction.

The coenzyme function of TPP analogues: 4'-NH-methyl-TPP,6'-methyl-TPP and 6'-methyl-4-nor-TPP has been studied in a transketolase-catalyzed reaction. Their dissociation constants have been found with the aid of the circular dichroism method, and coenzyme activity has been determined in a complete transketolase reaction, involving the substrate-donor and the substrate-acceptor, and also at the intermediate stage (by the alpha-carbanionic intermediate oxidation rate). The coenzyme activity values have been found different and largely dependend on the nature of the substrates used. A possibility of TPP functioning by the "two-center mechanism" in a transketolase-catalyzed reaction is discussed.

Kinetics↗
PubMed1984

Conformational specificity of chymotrypsin toward proline-containing substrates.

A number of peptide-4-nitroanilide substrates containing proline within the peptide chain have been synthesized and subjected to chymotryptic hydrolysis. Values of kcat and Km have been obtained from measurements at pH 7.8 and 25.0 degrees C. Kinetic studies at high enzyme concentrations up to 6.0 X 10(-4) mol X 1(-1) have allowed the evaluation of the conformational specificity of chymotrypsin due to the observation of various kinetic phases during the time-course of the reaction. When proline occupies the P2 position within the peptide chain, it is shown that the enzyme cleaves only the trans isomer of the substrate. The conformational specificity has also been studied for proline in P4 and P5 positions of the substrate. In some cases, an enzyme-catalyzed hydrolysis of the cis isomer was detected. From the amplitude ratios and the rate constants of the kinetic phases, information about the structural dependency of the cis/trans interconversion could be obtained. Charged residues N-terminal to the isomeric bond are of little influence on either cis/trans ratio or the rate of cis to trans interconversion. Extending the peptide chain N-terminal to the isomeric bond by alanine decreases to a low extent the cis content and increases the rate constant of the trans isomer formation.

Aniline Compounds↗
PubMed1983

A rapid procedure for the preparation of highly purified pyruvate decarboxylase from brewer's yeast.

A rapid purification procedure for pyruvate decarboxylase (E.C. 4.1.1.1.) from fresh cells of brewer's yeast (Saccharomyces carlsb.) is reported. The preparation of a crude enzyme (30-45 U/mg) by the use of fractionation steps with protamine sulfate, acetone, and ammonium sulfate takes about 6-7 h. A stable pyruvate decarboxylase (70-85 U/mg) was obtained from such preparations after purification on CM Sephadex C 50 after another 2-3 h. Stability and structural properties are compared for enzymes prepared from fresh and dried yeast.

Carboxy-Lyases↗
PubMed1982

[Degradation of inactivated alpha-amylase by associated proteases].

Alpha-Amylase preparations often contain small quantities of proteolytic activity which are difficult to remove. On the example of fungal alpha-amylase, such associated proteases have been shown to possess a specific activity to the denatured amylase molecules. The amylase is not attacked under native conditions, whereas in the thermal denaturation a rapid degradation of only the inactivated molecules occurs. A specific metabolic function of these associated proteases in the return of denatured amylase molecules to the amino acid pool is suggested.

Amylases↗
PubMed1980

Catalytic properties and electrostatic potential of charged immobilized enzyme derivatives. Pyruvate decarboxylase attached to cationic polystyrene beads of different charge densities.

Pyruvate decarboxylase has been covalently attached to positively charged macroporous polystyrene resins. The changes in the functional behaviour of the enzyme resulting from the electrostatic interaction of the cationic carrier polymer and the anionic substrate have been studied as function of the fixed charge concentration of the carrier. The findings confirm the connection of two widely used theoretical expressions relating electrostatic parameters with Michaelis constants.

Carboxy-Lyases↗
PubMed1980

[Relationship between catalytic properties, fixed charge concentration and electrostatic potential of polyelectrolyte-bound enzymes].

An equation for the calculation of the electrostatic potentials of polyelectrolyte-enzyme supports from electrostatic parameters has been derived by relating two different theories which describe the catalytic behaviour of polyelectrolyte-bound enzymes. The electrostatic potentials of polyionic supports have been determined by use of experimental results, on the one hand, from the fixed charge concentration and the ionic strength, on the other hand, from pH- and Km-shifts of immobilized enzymes. The accordance of potentials calculated from electrostatic and kinetic parameters confirms the macroscopic carrier-enzyme model.

Amylases↗
PubMed1979

Activation of fungal alpha-amylase by dithioerythritol.

The activity of fungal alpha-amylase has been shown to be influenced by disulfide-reducing reagents. Thus, the enzymatic activity increases in the presence of dithioerythritol or 2-mercaptoethanol. L-Cysteine is also capable of increasing the activity, but the activation competes with an inactivation reaction which dominates at higher reagent concentrations (greater than 20 mM). A possible scheme interpreting the results is given.

Amylases↗
PubMed1978

The mechanism of substrate activation of pyruvate decarboxylase: a first approach.

The sigmoidal shape of the curve for v[S], characteristic of pyruvate decarboxylase, indicates that the catalytic activity of this enzyme is regulated by the substrate. The enzyme, which is inactive in the absence of its substrate, is activated not only by 2-oxo acids but also by 2-oxo acid amides, which cannot act as a substrate of the enzyme. Whilst the dissociation constant of the enzyme-activator complex depends on the electrophilic nature of the carbonyl group, the catalytic activity reached at saturation concentrations of the activator species is independent of the structure of the activator molecules. The mechanism of activation which proceeds via two reversible steps could be evaluated exactly by stopped-flow techniques. The kinetic parameters of the activation and deactivation reaction were estimated and the validity of the equations derived which describe the activation kinetics could be proved by comparing them with the measured data. Using glyoxylic acid as an irreversibly binding active-site marker, it could be shown that addition of the substrate to the enzyme-bound thiamin diphosphate is the step of the catalytic mechanism whose rate is controlled by the substrate (activator) molecule.

Carboxy-Lyases↗
PubMed1978

[Effect of the chemical surface structure of functionalized polystyrene on the kinetic properties of immobilized yeast pyruvate decarboxylase].

The influence of the loading density of different functional groups and the length of suitable spacer structures on the kinetic properties of yeast pyruvate decarboxylase are investigated on identical polystyrene matrices. At constant concentrations of the fixed protein both the specific activity and the storage stability of the immobilized enzymes increase with increasing concentrations of the protein binding (C = O)-groups. pH-Optimum and K'M-value prove to be functions of the NH3+-content of the supports. Using four spacer resins with an equal content of spacer groups it could be shown that the optimum time of coupling as well as the maximum catalytical activity, storage stability and thermostability depend on the length of the spacer structures. On the other hand, the mobility of an ESR-marker fixed via the same spacers to the resin is not affected by the different spacer structures.

Carboxy-Lyases↗
PubMed1978

The influence of charged matrix surfaces on the thermostabilizing effect of calcium ions on immobilized fungal alpha-amylase.

The stabilizing effect of calcium ions on fungal alpha-amylase (EC 3.2.1.1) immobilized on a polystyrene anion exchanger (P+ amylase) was investigated and compared to the behaviour of soluble amylase. Moreover, gamma-(1,4-benzoquinone-2-yl)-aminopropyl silica-amylase (Si(n) amylase) as a conjugate with weakly basic amino groups and gamma-succinamidopropyl silica amylase (Si- amylase) as a conjugate with free carboxyl groups were applied for comparison. Depending on the calcium ion concentration the immobilized amylases showed a lower thermal stability than the soluble enzyme. The reduced stability was attributed to matrix effects in the microenvironment of the immobilized amylases and the calcium ion concentration in the carrier phase, which was changed in comparison with the external solution. Contrary to the non-measurable matrix effects in the microenvironment, altered calcium ion concentrations in the carrier phase of the polystyrene anion exchanger (P+) and gamma-succinamidopropyl silica (Si-) could be detected. With increasing calcium ion concentration a greater decrease of activity was observed for the soluble amylase than for the immobilized enzymes. The thermal stability of soluble amylase and P+ amylase was studied in dependence on pH. In the acidic pH-range P+ amylase indicated a higher thermal stability than the soluble enzyme in the presence of Ca2+ as well as in the absence of Ca2+. Contrary to soluble amylase the stabilizing effect of calcium ions on P+ amylase begins already at pH 3.5. Kinetic investigations for thermal inactivation were performed on soluble amylase and P+ amylase in the presence and absence of Ca2+ in the temperature range between 44--60 degrees C. Thermal inactivation proceeded by first order reactions. The inactivation rate constants kin served as a measure of thermal stability for discussing the stabilizing effect by Ca2+ depending on the temperature. The activation energies of inactivation EA were determined from the Arrhenius-plot of the inactivation rate constants.

Amylases↗