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A Schellenberger

Publications and source records attributed to A Schellenberger.

At least 19 recordsLinked to original sources
PubMed1998

Sixty years of thiamin diphosphate biochemistry.

The mechanism of ThDP enzymes originates in the anionic (ylid) structure of the coenzyme. On the other hand, no ylid species (as permanently existing structure) could be detected by 13C2-NMR studies with PDC (yeast), when the cofactor binds to the active site. Therefore, the rate of ylid formation as the first step of the catalytic mechanism distinguishes decisively the power (kcat) of all ThDP enzymes. 2H/1H-exchange experiments with PDC, TK, PDH and POX have shown that within the active center of ThDP enzymes (under native pH conditions!) the aminopyrimidine part generates the essential ylid structure by enhancing the dissociation rate (acidity) of the C2-H bond up to 4-6 orders of magnitude. Moreover, it could be proved that the mechanism of substrate activation of PDC (yeast) is also connected directly with the C2-H activation by the aminopyrimidine part. Experiments with analogs of ThDP or modified apoenzymes (via site-directed mutagenesis) have shown that this mechanism requires as essential elements a hydrogen bond between the pyrimidine N1' atom and a conserved Glu side chain of the different apoenzymes as well as the (evolutionary conserved) V-conformation. The latter positions the 4'-amino group in direct (functional) contact to the C2-H bond. A proposal is discussed, how the 4'-positioned amino group in cooperation with the N1' atom could increase the C2-H dissociation rate.

Catalysis↗
PubMed1995

The presence of a hydroxyl group at the C-1 atom of the transketolase substrate molecule is necessary for the enzyme to perform the transferase reaction.

Transketolase catalyzes the transfer of an aldehyde residue from keto sugars to aldo sugars. The intermediate product is dihydroxyethylthiamine pyrophosphate (DHETPP). In the absence of an acceptor substrate, the reaction is stopped at this stage and DHETPP does not undergo subsequent transformations. Pyruvate decarboxylase catalyses pyruvate decarboxylation to yield free aldehyde. The intermediate product is hydroxyethylthiamine pyrophosphate (HETPP). It differs from DHETPP only in that it has no hydroxyl at the C-2 atom of the aldehyde residue. We have shown that transketolase can bind HETPP and split the aldehyde residue from it. This fact suggests that the path of the reaction is determined by the absence (in HETPP) or presence (in DHETPP) of a hydroxyl group. In the former case the reaction will yield free aldehyde, in the latter the aldehyde residue will be transferred onto an acceptor substrate.

Apoenzymes↗
PubMed1994

Specificity of coenzyme binding in thiamin diphosphate-dependent enzymes. Crystal structures of yeast transketolase in complex with analogs of thiamin diphosphate.

The three-dimensional structures of complexes of yeast apotransketolase with the coenzyme analogs 6'-methyl, N1'-pyridyl, and N3'-pyridyl thiamin diphosphate, respectively, were determined with protein crystallographic methods. All three coenzyme analogs bind to the enzyme in a fashion highly similar to the cofactor thiamin diphosphate. Thus, either one of the hydrogen bonds of the pyrimidine ring nitrogens to the protein is sufficient for proper binding and positioning of the cofactor. The lack of catalytic activity of the N3'-pyridyl analog is not due to incorrect orientation of the pyrimidine ring, but results from the absence of the hydrogen bond between the N1' nitrogen atom and the conserved residue Glu418. The structure analysis provides further evidence for the importance of this conserved interaction for enzymatic thiamin catalysis.

Coenzymes↗
PubMed1993

The influence of the effectors of yeast pyruvate decarboxylase (PDC) on the conformation of the dimers and tetramers and their pH-dependent equilibrium.

The influence of effectors of yeast pyruvate decarboxylase, phosphate, pyruvamide, thiamin diphosphate and Mg++, on the pH-dependent equilibrium between dimers and tetramers was studied by synchrotron radiation X-ray solution scattering. Thiamin diphosphate and phosphate shift the equilibrium to higher pH values without altering the structure of the oligomers. Pyruvamide, a substrate analogue activator, induces a significant change in the structure of the tetramer. By eliminating radiation damage by addition of dithioerythrol to the buffers, the scattering curves could be measured accurately over a large angular range. They were expanded in terms of spherical harmonics to obtain the shapes of the dimers and tetramers with higher resolution than was hitherto possible. This also allowed us to position the dimers, which are centrosymmetric at low resolution, in the tetramers which have 222 symmetry. The results indicate that addition of pyruvamide results in a less compact tetramer owing to structural changes in the dimers and to their displacements.

Biophysical Phenomena↗
PubMed1993

Subsite affinities of Aspergillus niger glucoamylase II determined with p-nitrophenylmaltooligosaccharides.

Kinetic parameters were obtained for glucoamylase catalysed hydrolysis of substrates of an alpha-(1,4)-maltooligosaccharide series and of a p-nitro-phenyl-alpha-maltooligosaccharide series. p-Nitrophenyl substrates of chain length 11 and 17 were synthesized in 97% and 95% purity, respectively, to test the significance of binding at remote subsites. The affinities of the subsites > 4 are demonstrated to be insignificant. The subsite binding contributions for D-glucopyranosyl and for p-nitrophenyl residues were calculated.

Aspergillus niger↗
PubMed1993

Effects of metal ions, thiamine diphosphate analogues and subunit interactions on the reconstitution behaviour of pyruvate decarboxylase from brewer's yeast.

The reconstitution of pyruvate decarboxylase starts with reversible binding of thiamine diphosphate and Mg2(+)-ions to the apoenzyme, followed by a rate-limiting conformational change to the catalytically active holoenzyme. Investigations with diphospho-esters of 4-methyl-5-(2-hydroxyethyl)thiazolium derivatives have shown that the diphosphate residue of thiamine diphosphate is the most important part of the coenzyme responsible for the first reversible binding step. Methylation of the N1'-atom of the pyrimidine ring of thiamine diphosphate or 4'-oxythiamine diphosphate prevents the coenzyme from binding stably to the apoenzyme, so that the methylated coenzyme displays no coenzyme activity. In contrast, thiamine diphosphate analogues with bulky residues on the neighbouring C2'-atom of the pyrimidine ring form active holoenzyme complexes. This result shows the essential role of the N1'-atom of thiamine diphosphate in stable cofactor binding. The cofactor binding rate to the dimeric and tetrameric apoenzymes indicates that the cofactor is located in the contact regions of the subunits in the tetrameric enzyme.

Binding Sites↗
PubMed1992

Synchrotron radiation solution X-ray scattering study of the pH dependence of the quaternary structure of yeast pyruvate decarboxylase.

The pH dependence of the quaternary structure of pyruvate decarboxylase from yeast was studied in the range 6.2 less than pH less than 8.4. There is an equilibrium with a midpoint around pH 7.5 between tetramers and dimers, and the catalytic activity of the enzyme depends on the volume fraction of tetramer. This equilibrium may provide an additional regulating mechanism besides substrate activation since accumulation of pyruvate would lead to a reduction in pH and hence an increase of the concentration of the catalytically active tetramer. Radiation damage during the X-ray scattering experiments results in a shift of this equilibrium and in the formation of octamers. These effects could be circumvented and analyzed using experimental and data processing methods which can be readily applied to other radiation-sensitive systems. The low-resolution shapes of the dimers and tetramers were determined from the scattering curves using spherical harmonics. The results indicate that a conformational change must occur in the dimers upon formation of the tetramers, in agreement with earlier circular dichroism measurements.

Hydrogen-Ion Concentration↗
PubMed1992

Kinetic mechanism of pyruvate decarboxylase. Evidence for a specific protonation of the enzymic intermediate.

Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium. The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4-dinitrophenyl hydrazone. The protium content of 56% at acetaldehyde C1 demonstrates a specific protonation of the corresponding intermediate by the enzyme. Proton inventory studies and enzyme modification indicate the 4' amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor. A 'partially concerted' mechanism is suggested for the reaction steps following the decarboxylation.

Acetaldehyde↗
PubMed1992

Thiamin pyrophosphate binding mechanism and the function of the aminopyrimidine part.

Besides the pyrophosphate group, acting as the essential and primary binding function of TPP the N1-atom of the aminopyrimidine component functions as a second and also essential anchor to the protein component. Only if both of the contacts are formed the productive conformation of TPP within the active site of TPP enzymes is realized. A mechanism is proposed, which explains the results of our experiments with TPP-analogs.

Amines↗
PubMed1992

The catalytic power of pyruvate decarboxylase. A stochastic model for the molecular evolution of enzymes.

Pyruvate decarboxylase (PDC) catalyzes the decarboxylation of pyruvate anion by a factor of around 10(12), compared with the non-enzymic decarboxylation by thiamine, under standard state conditions of 1 mM pyruvate and thiamine diphosphate (TDP), pH 6.2. Free-energy diagrams constructed on the basis of earlier measurements for the enzymic and non-enzymic reactions give some information on catalysis by PDC. PDC stabilizes the reactant state preceding TDP addition to pyruvate by 76 kJ mol-1 and the transition state for the addition by 83 kJ mol-1. PDC stabilizes the reactant state preceding decarboxylation (presumably alpha-lactyl-TDP) by 27 kJ mol-1 and the transition state for decarboxylation by 68 kJ mol-1. In addition, the free-energy diagrams reveal a leveling of reactant-state free energies in the enzymic reaction compared with the non-enzymic reaction, in that the former are nearly equal to each other. The enzyme-bound transition-state energies are similarly leveled. The energetic leveling of reactant states has been noted by Albery, Knowles and their coworkers in many enzymic reactions and termed 'matched internal thermodynamics.' They showed that the result would arise naturally (and inevitably) in the 'evolution to perfection' of enzymes, when the evolutionary process was treated by a deterministic model. The critical assumption of this model was the validity of a Marcus-type or Brønsted-type linear free-energy relationship between rate and equilibrium constants for reactions occurring wholly within enzyme complexes. Here a completely stochastic simulation of molecular evolution, with no deterministic assumptions, is shown to reproduce both 'matched internal thermodynamics' and the 'matched internal kinetics' or leveling of transition-state energies noted here. The Albery-Knowles result is thus more general than might have been supposed.

Biological Evolution↗
PubMed1991

Immobilization of invertase by encapsulation in polyelectrolyte complexes.

Free and polystyrene-bound invertase from Saccharomyces cerevisiae were encapsulated within symplex membranes which were composed of cellulose sulfate as the polymeric anion and poly(dimethyldiallylammonium chloride) as the polymeric cation. The kinetics and the performance of the encapsulated enzyme preparations have been compared to the free enzyme employing the hydrolysis of sucrose. The pH and temperature optima were only slightly affected by the encapsulation. The kinetic constants, however, were changed by the encapsulation as a result of diffusional limitation. Encapsulated invertase showed a high storage stability and a high operational stability if low substrate concentrations were applied. The coimmobilization of invertase with living cells, which are not capable of utilizing sucrose, in the described capsules, opens many possibilities in fermentation technology.

Enzyme Stability↗
PubMed1990

An X-ray solution scattering study of the cofactor and activator induced structural changes in yeast pyruvate decarboxylase (PDC).

Structure and activation pattern of pyruvate decarboxylase (PDC) from yeast was studied by synchrotron radiation X-ray solution scattering. The results give a direct proof that the reversible deactivation of PDC at pH 8.0 is accompanied by the dissociation of the tetrameric holoenzyme into dimeric halves. The kinetics of this process was followed. At pH 6.5 the dimeric halves reassociate to a tetramer even in the absence of cofactors. The changes of the scattering pattern upon binding of the substrate-like activator pyruvamide indicate that the structure expands in the course of the enzyme activation.

Apoenzymes↗
PubMed1990

Cross-linking of pyruvate decarboxylase. Characterization of the native and substrate-activated enzyme states.

In order to demonstrate the role of the protein component of pyruvate decarboxylase in the mechanism of substrate activation, we have isolated and characterized two states of the enzyme, the non-activated and the substrate-activated state, by covalent linking with bifunctional reagents. Because of the fact that modification of the reactive amino groups by 2,4,6-trinitobenzenesulfonic acid or methyl propionimidate influences neither the catalytic nor the regulatory properties of pyruvate decarboxylase, we used bisimidates of different chain length in the modification experiments. Both the non-activated and the substrate-activated enzyme states could be characterized separately. The lag phase of product formation as a typical property of the native enzyme disappeared completely when the enzyme had been cross-linked in the presence of the substrate. The permanently activated enzyme state shows 85% of the activity of native pyruvate decarboxylase and is exclusively stabilized by intra-subunit links. Elimination and subsequent reincorporation of the cofactors thiamine pyrophosphate and magnesium ions resulted in a complete regaining of the properties of the permanently activated enzyme form. An inactive enzyme form was obtained after cross-linking of non-activated pyruvate decarboxylase at low ionic strength (less than 0.01). Using a disulfide-containing linker we could prove that the inactivity of the obtained enzyme preparation was only the result of the incorporated cross-links and not that of denaturation.

Cross-Linking Reagents↗
PubMed1989

Protein stabilization by blocking the native unfolding nucleus.

Studies on the thermal inactivation of immobilized enzymes result in a novel conception of protein stabilization. The native protein molecule is suggested to be characterized by a specific structural region where the unfolding process starts. Accordingly, enzyme stabilization by immobilization is the result of blocking this unfolding nucleus.

Amylases↗
PubMed1988

31P NMR investigations on free and enzyme bound thiamine pyrophosphate.

Pyruvate decarboxylase (PDC) contains thiamine pyrophosphate (TPP) and Mg2+ as cofactors. 31P NMR studies with PDC in the presence of added Mn2+ reveal the pyrophosphate moiety of TPP to be a nonaccessible area for the external Mn2+ and thus proving the Mg-P-complex (taking part in the binding of the coenzyme to the protein) to be a nonaccessible area for the medium. Glyoxylic acid, acting as an inhibitor of PDC by forming a noncleavable bond with the catalytic center of TPP causes a steric immobilization of the coenzyme indicated by a line broadening of the pyrophosphate moiety.

Carboxy-Lyases↗
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↗