Direct electron transfer from modified glassy carbon electrodes carrying covalently immobilised mediators to a dissolved viologen accepting pyridine nucleotide oxidoreductase and dihydrolipoamide dehydrogenase

N,N′-Diaminopropyl-4,4′-bipyridinium dication (DAPV) and cobalt diaminosarcophagine (Co—diamsar) were covalently bound on functionalised glassy carbon electrodes. The electron transfer from the modified electrodes to dissolved dihydrolipoamide dehydrogenase (Lip-DH) or viologen accepting pyridine nucleotide oxidoreductase (VAPOR) was tested by NADH formation in potentiostatically controlled electrochemical cells. The NADH formed was used to reduce pyruvate to -lactate catalysed by -lactate dehydrogenase ( -LDH). The reaction kinetics between the modified electrodes and the dissolved enzymes have been examined.     

Current mechanistic understanding of thiamin diphosphate-dependent enzymatic reactions

The mechanism of thiamin diphosphate-dependent enzymatic reactions is discussed, concentrating on two enzymes involved in decarboxylating pyruvic acid, the yeast pyruvate decarboxylase and the pyruvate dehydrogenase multienzyme complex from Escherichia coli. The availability of high-resolution X-ray structures for several thiamin diphosphate-dependent enzymes, the use of site-specifically substituted protein variants (resulting from site-directed mutagenesis), the development of model reactions for the various putative intermediates, and the application of new mechanistic tools in solution have all contributed to a much better understanding of the role of the protein component in catalysis. Perhaps the most important advance in our understanding of these mechanisms concerns the role of the 4'-aminopyrimidine component of the coenzyme, widely ignored prior to the publication of the X-ray results. The current view is that the two aromatic rings both contribute to catalysis, perhaps carrying out an intramolecular proton transfer to initiate the various reactions, an ability that makes this coenzyme virtually unique among coenzymes.     

Structural determinants for the efficient and specific interaction of thioredoxin with 2-oxoacid dehydrogenase complexes

Specificity and efficiency of thiored oxin action upon the 2-oxoacid dehydrogenase complexes are studied by using a number of thiored oxins and complexes. Bacterial and mammalian pyruvate and 2-oxoglutarate dehydrogenase systems display similar row of preference to thioredoxins that may result from thioredoxin binding to the homologous or common dihydrolipoamide dehydrogenase components of the complexes. The most sensitive tothioredoxin is the complex whose component exhibits the highest sequence similarity to eukaryotic thioredoxin reductase. Hence, thioredoxin binding to the complexes may be related to that in the thioredoxin reductase, a dihydrolipoamide dehydrogenase homolog. The highest potency of mitochondrial thioredoxin to affect the mitochondiral complexes is revealed. A 96–100% conservation of the mitochondrial thioredoxin structure is shown within the four known sequences and the N-terminus of the pigheart protein determined. Eleven thioredoxins tested biochemically are analyzed by multiple sequence alignment and homology modeling. Their effects correlate with the residues at the contact between the α 3/3₁₀ and α 1 helices, the length of the α 1 helix and charges in the α2–β3 and β4–β5 linkers. Polarization of the thioredoxin molecule and its active site surroundings are characterized. Thioredoxins with a highly polarized surface around the essential disulfide bridge (mitochondiral, pea f, and Arabidopsis thaliana h3) show low cross-reactivity as compared to the species with a decreased polarization of this area (e.g., from Escherichia coli). The strongest polarization of the whole molecule results in the highest magnitude of the electrostatic dipole vector of mitochondrial thioredoxin. Thiored oxins with the dipole orientation similar to that of the latter have the affinities for the 2-oxoacid dehydrogenase complexes, proportional to the dipole magnitudes. Thioredoxin with an opposite dipole orientation shows no effect. Activating and inhibitory thioredoxin disulfides are distinguished by the charges of the residues 13/14 (α1 helix(, 51 (α2–β3 linker), and 83/85 (β4–β5 linker), changing the dipole direction. The results show that the thioredoxin-target interplay may be controlled by the long-range interactions between the electrostatic dipole vectors of the proteins and the degree of their interface polarization.     

Antiglioma pseurotin A from marine Bacillus sp. FS8D regulating tumour metabolic enzymes

Pseurotin A was isolated from a culture of marine Bacillus sp. FS8D and showed to be active against the proliferation of four different glioma cells with IC₅₀ values of 0.51–29.3 μM. It has been found that pseurotin A downregulated the expression of tumour glycolytic enzymes pyruvate kinase M2 (PKM2) and lactate dehydrogenase 5 (LDH5) and upregulated the expression of pyruvate dehydrogenase beta (PDHB), adenosine triphosphate synthase beta (ATPB) and cytochrome C (Cyto-C), the important regulators for tricarboxylic acid cycle and oxidative phosphorylation. The data suggested that targeting multiple metabolic enzymes might be one of the antiglioma mechanisms of pseurotin A.     

Changes in the brain protein levels following administration of kainic acid

Kainic acid (KA), a potent neurotoxin and excitatory amino acid, leads to derangements and modulation of brain proteins. No global brain protein expression pattern induced by KA-treatment has been reported yet. We therefore studied the effect of systemic KA administration on the levels of brain proteins. Rats were injected placebo or KA intraperitoneally and brain was taken after one week. The mitochondrial and cytosolic fractions of the brain proteins were analyzed by proteomics technologies and the levels of selected proteins were quantified using specific software. Heat shock protein HSP 27 was exclusively detected in brains of animals treated with KA, whereas the glucose regulated protein GRP 78 was downregulated. The levels of neurofilaments and alpha-internexin were significantly decreased and a fragment of tubulin alpha-1 chain was manifold increased in KA-brains. The mitochondrial enzymes dihydrolipoamide dehydrogenase, ATP synthase beta chain and isocitrate dehydrogenase were reduced and pyruvate kinase M1 was increased following KA treatment. We conclude that the concomitant determination of the brain proteins indicates altered regulation of heat shock proteins, neuronal death, cytoskeletal disruption, and mitochondrial derangement by systemic KA administration. This report confirms and extends previous studies on the effect of KA on the expression of brain proteins and suggests that our analytical system can serve as a model for neurotoxicological, neurobiological, and neuropathological proteome studies.     

A replaceable dual-enzyme capillary microreactor using magnetic beads and its application for simultaneous detection of acetaldehyde and pyruvate

A novel replaceable dual-enzyme capillary microreactor was developed and evaluated using magnetic fields to immobilize the alcohol dehydrogenase (ADH)- and lactate dehydrogenase (LDH)-coated magnetic beads at desired positions in the capillary. The dual-enzyme assay was achieved by measuring the two consumption peaks of the coenzyme β-nicotinamide adenine dinucleotide (NADH), which were related to the ADH reaction and LDH reaction. The dual-enzyme capillary microreactor was constructed using magnetic beads without any modification of the inner surface of the capillary, and showed great stability and reproducibility. The electrophoretic resolution for different analytes can be easily controlled by altering the relative distance of different enzyme-coated magnetic beads. The apparent K(m) values for acetaldehyde with ADH-catalyzed reaction and for pyruvate with LDH-catalyzed reaction were determined. The detection limits for acetaldehyde and pyruvate determination are 0.01 and 0.016 mM (S/N = 3), respectively. The proposed method was successfully applied to simultaneously determine the acetaldehyde and pyruvate contents in beer samples. The results indicated that combing magnetic beads with CE is of great value to perform replaceable and controllable multienzyme capillary microreactor for investigation of a series of enzyme reactions and determination of multisubstrates.     

Application of high-performance liquid chromatography to the purification, disintegration and molecular mass determination of pyruvate dehydrogenase multi-enzyme complexes from different sources

The pyruvate dehydrogenase complex is associated with the inner mitochondrial membrane. A gentle and rapid purification procedure, especially for the very unstable pyruvate dehydrogenase complex from the extremely thermophilic organism Thermus aquaticus, is described. This procedure is based essentially on a combination of hydrophobic interaction and of adsorption chromatography by the rapid fast protein liquid chromatographic technique. Applying the same method, a relative molecular mass of 9.1 . 10(6) daltons was obtained by gel filtration on Superose 6 HR 10/30 for the pyruvate dehydrogenase complex from T. aquaticus. The same column served to resolve the pyruvate dehydrogenase complex into its enzyme components.     

Determination of lipoyllysine derived from enzymes by liquid chromatography

Lipoyllysine was liberated from the commercial enzymes bovine heart alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase. After incubation of the enzymes with pronase for 4 h, the lipoyllysine liberated was determined by high-performance liquid chromatography with ultraviolet detection at 340 nm. Standard lipoyllysine was synthesized in our laboratory. The specific determination of lipoyllysine with ultraviolet detection only at 340 nm could be utilized for the enzyme hydrolysate samples. Recoveries of lipoyllysine added (5.0 micrograms) to a reaction mixture containing protease and bovine serum albumin or ovalbumin model proteins (1.0 mg) were 116.8 and 119.5%, respectively. The lipoyllysine content in beef heart alpha-ketoglutarate dehydrogenase was 0.55 microgram/mg of enzyme and 0.83 microgram/mg of enzyme protein in beef heart pyruvate dehydrogenase.     

Capillary electrophoresis-integrated immobilized enzyme microreactor with graphene oxide as support: Immobilization of negatively charged L-lactate dehydrogenase via hydrophobic interactions

We report the first application of hydrophobic interaction between graphene oxide (GO) and negatively charged enzymes to fabricate CE-integrated immobilized enzyme microreactors (IMERs) by a simple and reliable immobilization procedure based on layer by layer assembly. L -lactate dehydrogenase (L -LDH), which is negatively charged during the enzymatic reaction, is selected as the model enzyme. Various spectroscopic techniques, including SEM, FTIR, and UV-vis are used to characterize the fabricated CE-IMERs, demonstrating the successful immobilization of enzymes on the negatively charged GO layer in the capillary surface. The IMER exhibits excellent repeatability with RSDs of inter-day and batch-to-batch less than 3.49 and 6.37%, respectively, and the activity of immobilized enzymes remains about 90% after five-day usage. The measured K_m values of pyruvate and NADH of the immobilized L -LDH are in good agreement with those obtained by free enzymes. The results demonstrate that the hydrophobic interactions and/or π-π stacking is significant between the GO backbone and the aromatic residues of L -LDH and favorable to fabrication of CE-integrated IMERs. Finally, the method is successfully applied to the determination of pyruvate in beer samples.     

Kinetic studies and analytical application of cholesterol oxidase and peroxidase immobilized to synthetic polymer

A method for individual and simultaneous covalent immobilization of cholesterol oxidase and peroxidase to copolymer of acrylonitrile with acrylamide is described. The effect of immobilization on the catalytic properties of the covalently bound enzymes was studied. The immobilized enzymes showed no change in pH optima and an increase in temperature optima, activation energy, and K _m , compared to data received from experiments with soluble enzymes. A small glass column packed with immobilized multienzyme complex was used to develop a method for manual determination of cholesterol in foodstuffs (e.g., in mayonnaise “Olinease”). The method was characterized by high analytical precision (coefficient of variation = 2.67%). The results show high correlation with those obtained by the Kageyama method (r=0.986). The method is economical (the enzyme-carrier conjugate may be used more than 300 times), precise, easy to perform, and less time-consuming than the manual methods utilizing soluble enzymes. The established manual method can be proposed for cholesterol determination in foodstuffs.     

A Biosensor for ADP with Internal Substrate Amplification

Abstract

An enzyme electrode was constructed from an oxygen electrode and a layer incorporating four enzyme systems for the sensitive determination of ADP and ATP. The cofactor is cycled between pyruvate kinase and hexokinase under formation of pyruvate which is detected by the coimmobilized, sequentially acting enzymes lactate dehydrogenase and lactate monooxygenase. The enzymatic recycling results in a 220-fold increased sensitivity to ADP compared to the unamplified reaction.     

The relationship between growth enhancement andpet expression inEscherichia coli

Applied Biochemistry and Biotechnology - Thepet operon consists of genes coding for enzymes responsible for ethanol production and consists of pyruvate dehydrogenase and alcohol dehydrogenase II...     

Principles of multienzyme purifications by affinity chromatography

Recently in our laboratory, up to 20 different enzymes and their genetic variants have been purified from mouse andDrosophila by affinity chromatography. By virtue of the specific coenzyme requirements, up to ten different enzymes could be copurified from a single tissue extract either by biospecific elutions with different coenzymes or inhibitors, or by sequential passages of the extract through several cofactor-related affinity columns. Important principles were developed to purify enzymes exhibiting low affinity to the affinity columns. By “affinity filtration” of the extract through the affinity column, enzymes of low affinity can be retarded and separated effectively from strongly bound and nonadsorbed proteins. By the “saturation readsorption” procedure, enzymes of low affinity could be effectively separated from those of high affinity by overloading of the extracts on the affinity columns. Readsorption of the leaked low affinity enzymes to a second affinity column often results in better enzyme purification because of the elimination of competitive high affinity enzymes. With the application of these principles, the following enzymes and their genetic variants were highly purified via a single- or two-step affinity column procedure: lactate dehydrogenase-A, lactate dehydrogenase-B, lactate dehydrogenase-X, phosphoglycerate kinase-A, phosphoglycerate kinase-B, cytoplasmic and mitochondrial isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase, glutathione reductase, phosphoglucose isomerase and pyruvate kinase from mouse tissues; alcohol dehydrogenase, malate dehydrogenase, α-glycerol-phosphate dehydrogenase, malic enzyme, and glucose-6-phosphate dehydrogenase fromDrosophila.     

Lipid-polyamide—Polyethyleneimine microcapsules for immobilization of free cofactors and multienzyme system

Lipid-polyamide membrane microcapsules containing multienzyme system convert ammonia into glutamate.l-Glutamic dehydrogenase, alcohol dehydrogenase, NAD, and alpha-ketoglutarate retained within the microcapsules do not leak out. This way the multienzyme system can recycle the free cofactor NAD and synthesize glutamate from external lipophilic ammonia diffusing into the microcapsules. By using polyethyleneimine instead of hemoglobin as microcapsule filler it is possible to obtain higher conversion rates and to increase the storage stability of the recycling activity.     

Immobilization of Enzymes on Polychlorotrifluoroethylene Particles Packed in Small Columns

Abstract

The use of polychlorotrifluoroethylene (PCTFE) particles packed in 5–10 cm × 0.4 cm ID columns for the immobilization of enzymes by hydrophobic adsorption is investigated. Enzyme binding capacity of PCTFE is about 0.2 mg/gm of polymer. PCTFE-immobilized lactate dehydrogenase, alcohol dehydrogenase, and urease-glutamate dehydrogenase are demonstrated to be useful for the determinations of pyruvate, ethanol, and urea, respectively. Immobilized enzyme lifetimes are generally about 1–2 months.     

含磷丙酮酸脱氢酶系抑制剂的研究进展

丙酮酸脱氢酶系是生物体内重要的氧化还原酶之一,其催化丙酮酸氧化脱羧转化为乙酰辅酶A是生物体内的关键代谢过程,在生物化学研究中具有重要的意义.根据生物化学的理由来进行新药分子生物合理设计的观点,丙酮酸脱氢酶系是一个具有重要意义和值得探索的靶标.研究表明许多有机磷化合物是一类重要的丙酮酸脱氢酶系抑制剂.本文系统介绍了含磷丙酮酸脱氢酶系抑制剂的研究历程和研究动态,并结合本课题组的研究工作对代表性的研究结果和进展进行了总结和评述.     

Antimicrobial activity of rhodomyrtone isolated from Rhodomyrtus tomentosa (Aiton) Hassk

Abstract

Rhodomyrtone was isolated from the leaves of Rhodomyrtus tomentosa (Aiton) Hassk grown in Vietnam using chromatographic methods. Its chemical structure was confirmed by means of spectroscopic data analysis. The pH drop measurement, enzyme activity assays and fluorescence stain were used to examine rhodomyrtone anticaries activity. It was found that rhodomyrtone suppressed acid production by Streptococcus mutans, a major cariogenic agent in human by inhibiting enzyme activities responsible for acid production and tolerance, including membrane bound enzymes F-ATPase and phosphotransferase system (PTS), as well as glycolysis enzymes glyceraldehyphosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) in cytoplasm with the IC₅₀ values of 24?μM, 19?μM, 23?μM and 28?μM, respectively. Moreover, 50?μM rhodomyrtone reduced biofilm biomass formed by S. mutans up to 59% (p?<?0.05). Fluorescent images indicated that cells on the biofilms were significantly killed. Thus, rhodomyrtone is a new and potential anticaries agent against S. mutans.     

Central Carbon Metabolism in Marine Bacteria Examined with a Simplified Assay for Dehydrogenases

A simplified assay platform was developed to measure the activities of the key oxidoreductases in central carbon metabolism of various marine bacteria. Based on microplate assay, the platform was low-cost and simplified by unifying the reaction conditions of enzymes including temperature, buffers, and ionic strength. The central carbon metabolism of 16 marine bacteria, involving Pseudomonas, Exiguobacterium, Marinobacter, Citreicella, and Novosphingobium were studied. Six key oxidoreductases of central carbon metabolism, glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, 2-ketoglutarate dehydrogenase, malate dehydrogenase, malic enzyme, and isocitrate dehydrogenase were investigated by testing their activities in the pathway. High activity of malate dehydrogenase was found in Citreicella marina, and the specific activity achieved 22 U/mg in cell crude extract. The results also suggested that there was a considerable variability on key enzymes’ activities of central carbon metabolism in some strains which have close evolutionary relationship while they adapted to the requirements of the niche they (try to) occupy.     

Determination of the binding specificity of the 12S subunit of the transcarboxylase by saturation transfer difference NMR

In this study we present the characterization of the interaction of biotin and methylmalonyl-CoA (MMCoA) with the carboxyltransferase subunit (12S) from the transcarboxylase (TC) from Propionibacterium shermanii. This biotin dependent multienzyme complex catalyses the transfer of carbon dioxide from methylmalonyl-CoA (MMCoA) to pyruvate. The Saturation Transfer Difference NMR (STD) technique was performed to determine the binding epitope from biotin and MMCoA to the 12S subunit. We could show by titrations during STD experiments that biotin and MMCoA bind cooperatively in one binding pocket.     

Mass spectrometry in the study of extracellular enzymes produced by filamentous fungi

The use of MALDI-TOF and other types of mass spectrometry for the identification and investigation of extracellular enzymes (carbohydrases, proteinases, esterases, etc.) produced by filamentous fungi belonging to the genera Aspergillus, Trichoderma, Penicilium, Chrysosporium, etc. is discussed. The method of mass spectrometric peptide fingerprinting combined with the use of Internet software for on line data analysis (MASCOT, Aldente, FindPept, FindMod, GlycoMod) allows the fast and reliable identification of both individual enzymes and the components of crude multienzyme preparations without their fractionation. The method was also applied to the discrimination of the fungal genes encoding enzymes with similar substrate specificity. Other enzyme-based applications of mass spectrometry, such as the revelation of the presence or absence of structural domains in the molecules of carbohydrases, the detection of posttranslational and artificial modifications in the enzymes, and the use of tandem mass spectrometry for de novo peptide sequencing are described.