Kinetics Of Substrate Oxidation Research Articles

Measuring the short-term substrate utilization response to high-carbohydrate and high-fat meals in the whole-body indirect calorimeter.

The paper demonstrates that minute‐to‐minute metabolic response to meals with different macronutrient content can be measured and discerned in the whole‐body indirect calorimeter. The ability to discriminate between high‐carbohydrate and high‐fat meals is achieved by applying a modified regularization technique with additional constraints imposed on oxygen consumption rate. These additional constraints reduce the differences in accuracy between the oxygen and carbon dioxide analyzers. The modified technique was applied to 63 calorimeter sessions that were each 24 h long. The data were collected from 16 healthy volunteers (eight males, eight females, aged 22–35 years). Each volunteer performed four 24‐h long calorimeter sessions. At each session, they received one of four treatment combinations involving exercise (high or low intensity) and diet (a high‐fat or high‐carbohydrate shake for lunch). One volunteer did not complete all four assignments, which brought the total number of sessions to 63 instead of 64. During the 24‐h stay in the calorimeter, subjects wore a continuous glucose monitoring system, which was used as a benchmark for subject's postprandial glycemic response. The minute‐by‐minute respiratory exchange ratio (RER) data showed excellent agreement with concurrent subcutaneous glucose concentrations in postprandial state. The averaged minute‐to‐minute RER response to the high‐carbohydrate shake was significantly different from the response to high‐fat shake. Also, postprandial RER slopes were significantly different for two dietary treatments. The results show that whole‐body respiration calorimeters can be utilized as tools to study short‐term kinetics of substrate oxidation in humans.

Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study.

Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H2O2-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (kcat> 200 s−1) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 kcat ~20 s−1) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant.

The influence of hydroquinone on tyrosinase kinetics

In vitro studies, using combined spectrophotometry and oximetry together with hplc/ms examination of the products of tyrosinase action demonstrate that hydroquinone is not a primary substrate for the enzyme but is vicariously oxidised by a redox exchange mechanism in the presence of either catechol, l-3,4-dihydroxyphenylalanine or 4-ethylphenol. Secondary addition products formed in the presence of hydroquinone are shown to stimulate, rather than inhibit, the kinetics of substrate oxidation.

Microstructure and oxidation resistance of magnetron-sputtered nanocrystalline NiCoCrAlY coatings on nickel-based superalloy

Nanocrystalline NiCoCrAlY coatings on GH3128 nickel-based superalloy were produced using DC magnetron sputter deposition with a composite target. The microstructure and morphology of coatings were investigated using X-ray diffraction (XRD), scanning electron microscope (SEM), energy dispersive X-ray analysis (EDAX) and atomic force microscopy (AFM). Isothermal-oxidation tests were performed at 1100 °C in air for 24 h. The results show that the magnetron-sputtered NiCoCrAlY coating possesses a polycrystalline austenitic structure (FCC) with lattice constant 0.359821 nm. The (2 2 0) is the preferred growth direction of the NiCoCrAlY coating and the coating grain size is about 70 nm. The isothermal-oxidation test results exhibit that the oxidation kinetics of substrate and coating specimens all basically follow the parabolic rate law. However, the weight gain rate constant of coating is only about one-fifth of that of substrate. The weight gain of NiCoCrAlY coated specimen is only 48.16% of the superalloy substrate specimen after the isothermal-oxidation test. The above results reveal that the magnetron-sputtered nanocrystalline NiCoCrAlY coating is quite effective to increase the high temperature oxidation resistance of GH3128 superalloy.

Kinetics of substrate oxidation and hydrogen peroxide production by Mycoplasma mycoides subsp. mycoides Large Colony (LC) type and Mycoplasma mycoides subsp. capri

Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients ( K s) varied for different substrates. Substrate utilization patterns and K s values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.

Liquid-phase oxidation of benzothiophene and dibenzothiophene by cumyl hydroperoxide in the presence of catalysts based on supported metal oxides

The liquid-phase oxidation of benzothiophene and dibenzothiophene by cumyl hydroperoxide in the presence of supported metal oxide catalysts was carried out in octane in an N2 atmosphere at 50–80°C. The cumyl hydroperoxide, benzothiophene, and dibenzothiophene conversions and the yield of sulfones were determined for catalysts of various natures. In the presence of MoO3/SiO2, the most efficient and most readily regenerable catalyst, the benzothiophene conversion was ∼60% and the dibenzothiophene conversion was as high as 100% upon almost complete consumption of cumyl hydroperoxide. The influence of unsaturated and aromatic compounds (oct-1-ene, toluene) on the catalytic effect was studied. The kinetics of substrate oxidation and cumyl hydroperoxide decomposition and an analysis of the cumyl hydroperoxide conversion products suggested a benzothiophene and dibenzothiophene oxidation mechanism including the formation of an intermediate complex of the hydroperoxide with the catalyst and the substrate and its transformation via heterolytic and homolytic routes.

Functioning of oxidative phosphorylation in liver mitochondria of high-fat diet fed rats

We proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24 ± 6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker N ε-(carboxymethyl)lysine. HFD did not significantly affect mitochondrial copy number, oxygen uptake, membrane potential (Δ ψ), ADP/O ratio, and the content of coenzyme Q 9, cytochromes b and a + a 3. Modular kinetic analysis showed that the kinetics of substrate oxidation, phosphorylation, proton leak, ATP-production and ATP-consumption were not influenced significantly. After HFD-feeding ANT exerted considerable control over oxygen uptake (control coefficient C = 0.14) and phosphorylation fluxes ( C = 0.15), extra- ( C = 0.23) and intramitochondrial ( C = − 0.56) ATP/ADP ratios, and Δ ψ ( C = − 0.11). We conclude that although HFD induces accumulation of LCAC and N ε-(carboxymethyl)lysine, oxidative phosphorylation does not adapt to these metabolic challenges. Furthermore, ANT retains control of fluxes and intermediates, making inhibition of this enzyme a more probable link between obesity and type 2 diabetes.

Biochemical characterisation of some non fermenting, non arginine hydrolysing mycoplasmas of ruminants

The pattern and kinetics of substrate oxidation by type and recent field strains of Mycoplasma agalactiae, Mycoplasma bovis, Mycoplasma bovigenitalium and Mycoplasma ovine/caprine serogroup 11 were investigated by measurement of oxygen uptake. Metabolism of a range of organic acids, sugars and alcohols was detected. All the test strains were unable to oxidise sugars, glycerol and the organic acids, fumarate, malate and alpha-ketoglutarate (1 mM). All strains oxidised organic acid l-lactate, 2-oxobutyrate and pyruvate and demonstrated the ability to oxidise alcohols, particularly isopropanol, which was oxidised at a high rate and high affinity (0.5 mol/mol isopropanol). Its oxidation was consistent with acetone formation, which may be of important in relation to pathogenicity. All strains oxidised similar substrates, however differences were observed between strains in terms of the relative rates and kinetic values for some substrates.

The pattern and kinetics of substrate metabolism of Campylobacter jejuni and Campylobacter coli.

The main aim was to investigate the patterns and kinetics of substrate oxidation by Campylobacter jejuni and C. coli. Substrate oxidation profiles by 100 strains were determined using oxygen electrode system. All the isolates tested oxidized formate, l-lactate, cysteine, glutamine and serine with high oxidation rates and high affinity but varied in their ability to oxidize citric acid cycle intermediates, aspartic acid and serine. Based on the oxidation ability of alpha-ketoglutarate, succinate, fumarate and aspartic acid, Campylobacter strains tested were divided into three distinct metabolic categories. The first group was able to metabolize alpha-ketoglutarate, succinate, fumarate and aspartic acid; the second group was unable to oxidize alpha-ketoglutarate; and the third group was unable to oxidize, succinate, fumarate, and aspartic acid. Furthermore, serine oxidation rate enabled the differentiation of C. jejuni and C. coli. Overall, the results highlights the extensive metabolic diversity between and within Campylobacter species. In addition, the kinetic data of oxidized substrates obtained may improve the isolation procedures of the organism.

Spectroscopic studies on human serum albumin and methemalbumin: optical, steady-state, and picosecond time-resolved fluorescence studies, and kinetics of substrate oxidation by methemalbumin.

The nature of the heme environment in methemalbumin, the Fe(III) protoporphyrin IX (heme)-human serum albumin (HSA) complex, was investigated by optical spectroscopy. Comparison of the optical spectra of methemalbumin, ferro-hemalbumin in the absence and presence of 2-methylimidazole, and their carbon monoxide derivatives with horseradish peroxidase (HRP) and its corresponding derivatives indicates that histidine is not present in the first coordination sphere of heme in methemalbumin and that the protein is devoid of a well-defined heme cavity. The complex exhibits peroxidase activity by catalyzing oxidation of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) by hydrogen peroxide. Its activity ( K(M)=433 microM, molar catalytic activity=0.33 s(-1)), however, is considerably lower compared to HRP, indicating differences in the heme environments. Fluorescence intensity decays of Trp214 in HSA and methemalbumin, best fitted to a three-exponential model, gave the lifetimes 7.03 ns (30%), 3.17 ns (38%), and 0.68 ns (32%) for HSA and 8.04 ns (1.7%), 2.42 ns (19.7%), and 0.64 ns (78.6%) for methemalbumin. These lifetime values were further confirmed by a model-independent maximum entropy method. Similarity in the lifetimes and variations in the amplitudes suggest that while conformational heterogeneity of HSA is unperturbed on heme binding, redistribution of the populations of the three conformations occurs and the sub-state associated with the shortest lifetime dominates the total population by approximately 80%. Decay associated spectra (DAS) indicate that the observed lifetime variation with wavelength is predominantly due to ground state heterogeneity, though solvent dipolar relaxation also contributes. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information on motion within the protein together with the whole protein molecule. The binding of heme did not affect the rotational correlation time of the albumin molecule (approximately 20 ns). However, the motion of tryptophan within the protein matrix increased by a factor of approximately 3 (0.46 ns to 0.15 ns). This indicates that while the overall hydrodynamic volume of the albumin molecule remained the same, tryptophan underwent a more rapid internal rotation because of the efficient energy transfer to the bound heme. Optical studies, analysis of lifetime measurements, DAS, and anisotropy measurements together suggest that heme binds to a surface residue. The rapid internal motion of Trp214 during its excited state lifetime for the approximately 80% populated conformer of methemalbumin allows the orientation factor, kappa(2), to approach the average value of 2/3. From the time-resolved fluorescence measurements and the energy transfer calculations on methemalbumin, a Trp214-heme distance of 22 A was deduced.

Primary causes of decreased mitochondrial oxygen consumption during metabolic depression in snail cells

Cells isolated from the hepatopancreas of estivating snails (Helix aspersa) have strongly depressed mitochondrial respiration compared with controls. Mitochondrial respiration was divided into substrate oxidation (which produces the mitochondrial membrane potential) and ATP turnover and proton leak (which consume it). The activity of substrate oxidation (and probably ATP turnover) decreased, whereas the activity of proton leak remained constant in estivation. These primary changes resulted in a lower mitochondrial membrane potential in hepatopancreas cells from estivating compared with active snails, leading to secondary decreases in respiration to drive ATP turnover and proton leak. The respiration to drive ATP turnover and proton leak decreased in proportion to the overall decrease in mitochondrial respiration, so that the amount of ATP turned over per O2 consumed remained relatively constant and aerobic efficiency was maintained in this hypometabolic state. At least 75% of the total response of mitochondrial respiration to estivation was caused by primary changes in the kinetics of substrate oxidation, with only 25% or less of the response occurring through primary effects on ATP turnover.

Kinetics of substrate oxidation by whole cells and cell membranes ofHelicobacter pylori

Oxygen uptake by Helicobacter pylori cells and membranes was determined. Cells from stirred broth cultures or agar plates, suspended in buffer, possessed a variable and apparently endogenous respiration which could be sustained for several hours. In contrast, oxygen consumption by cells from statically incubated broth cultures, in the absence of added substrate, was transient or undetectable. These latter cells, however, oxidised ethanol, fumarate, glucose, D-lactate, pyruvate and succinate, though glucose-oxidising ability declined rapidly. The Kms for D-lactate, pyruvate and succinate metabolism were low (< or = 20 microM) and oxygen uptake was approximately 1.5, 2 and 2 mol per mol substrate respectively, indicating metabolism beyond acetate plus CO2 and implying the presence of tricarboxylic acid cycle activity. Cell membranes oxidised fumarate, D-lactate, NADH, NADPH and succinate. NADPH oxidation was six times more rapid than that of NADH. Rates of oxygen uptake by cells suspended in buffer with metabolisable substrate were < 20% of those for cells suspended in a brain heart infusion medium. Uninoculated medium consumed significant quantities of oxygen.

Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori

Oxygen uptake by Helicobacter pylori cells and membranes was determined. Cells from stirred broth cultures or agar plates, suspended in buffer, possessed a variable and apparently endogenous respiration which could be sustained for several hours. In contrast, oxygen consumption by cells from statically incubated broth cultures, in the absence of added substrate, was transient or undetectable. These latter cells, however, oxidised ethanol, fumarate, glucose, d-lactate, pyruvate and succinate, though glucose-oxidising ability declined rapidly. The Kms for d-lactate, pyruvate and succinate metabolism were low (≤20 μM) and oxygen uptake was approximately 1.5, 2 and 2 mol per mol substrate respectively, indicating metabolism beyond acetate plus CO2 and implying the presence of tricarboxylic acid cycle activity. Cell membranes oxidised fumarate, d-lactate, NADH, NADPH and succinate. NADPH oxidation was six times more rapid than that of NADH. Rates of oxygen uptake by cells suspended in buffer with metabolisable substrate were < 20% of those for cells suspended in a brain heart infusion medium. Uninoculated medium consumed significant quantities of oxygen.

Effects and degradation of 4-chloro-3-cresol in a system of natural self-purification

For many chemical compounds there is much information about their toxicity and biodegradability, but hardly any ecotoxicological investigations take into consideration how xenobiotic substances are integrated into complex biological systems. Here it is shown that 4-chloro-3-cresol (CMC), a chlorinated phenolic derivative used as a disinfectant and preservative, is completely eliminated within the process of natural self-purification, imitated in a lab-scaled river model. Biodegration of CMC occurred after the elimination of other organic compounds (synthetic sewage) which were easier to degrade. The nitrification of ammonia only started after CMC had been completely degraded. The kinetics of substrate oxidation (acetate) measured by oxygen consumption (Warburg-method) were inhibited noncompetitively by CMC. The gradual increase of the CMC concentration in the influent of the plant led to an increasing insensitiveness of the bacterial biocoenosis so that the kinetics of substrate oxidation in the presence of CMC resulted in a decrease of the inhibitory effect (increasing inhibition constant K i). The rate of CMC degradation referring to the biomass concentration increased with increasing CMC concentration in the influent, which indicated a growth of CMC-degrading bacteria within the total population. CMC degradation itself was inhibited at high concentrations (“substrate surplus inhibition”). In addition higher CMC concentrations in the influent of the river model led to a change of (1) the bacterial community and (2) the diversity of ciliates populations. In practice the separation of the processes of the elimination of easy biodegradable substances and of the degradation of CMC (e.g. activated sludge reactor and trickling filter) should be considered.

Isolation and Physiological Characterization of Autotrophic Sulphur Bacteria Oxidizing Dimethyl Disulphide as Sole Source of Energy

The isolation of a number of strains of bacteria able to grow on dimethyl disulphide and dimethyl sulphide as sole source of energy is described. The isolates came from diverse habitats, including soil, peat, marine mud and a freshwater pond. The isolates were morphologically and physiologically best described as thiobacilli, capable of growth as Calvin cycle autotrophs on inorganic sulphur compounds, methylated sulphides or thiocyanate. They could not grow heterotrophically or methylotrophically. One isolate (E6) was examined in detail. Substrate oxidation kinetics indicated that methanethiol, sulphide, formaldehyde and formate, but not dimethyl sulphide, could be implicated as intermediates in dimethyl disulphide metabolism. Apparent K s values for the oxidation of dimethyl disulphide and methanethiol were 2.5 and 3·2 μm respectively. Growth yields in chemostat culture on dimethyl disulphide with and without thiosulphate indicated that energy conservation was probably coupled to the oxidation of formaldehyde and sulphide (derived from dimethyl disulphide via methanethiol) to CO2 and sulphate. Maximum growth yield (Y max) on dimethyl disulphide was 17 g cell-carbon per mol of dimethyl disulphide. At one dilution rate (0·078 h−1), the biomass of a culture limited by dimethyl disulphide increased when thiosulphate was also supplied, indicating a thiosulphate-dependent yield of 2·45 g cell-carbon mol−1. This is the first demonstration of the isolation of organisms into pure culture that are capable of growth on dimethyl disulphide as sole energy substrate, and of degrading it completely to CO2 and sulphate.

Oxidation of NADH and reduction of vitamin K 3 at the octane|water interface with ethioporphyrin complexes as catalysts

The redox reaction at the interface of two immiscible liquids between the reaction components in different phases and with ethioporphyrin metal complexes as catalysts has been investigated by measuring the Volta-potential and by spectroscopy. Co 3+- and Fe 3+-ethioporphyrins were found to be active catalysts, while metal free ethioporphyrins did not display catalytic properties. The oxidation kinetics of substrate in the aqueous phase was studied and the reduced forms of electron acceptors—vitamin K 3 and 1,4-naphthoquinone-were recorded. The interaction of a metal complex of porphyrins with water was also studied.

Oxidation of nadh and reduction of vitamin K 3 at the octane/water interface with ethioporphyrin complexes as catalysts

Abstract The redox reaction at the interface of two immiscible liquids between the reaction components in different phases and with ethioporphyrin metal complexes as catalysts has been investigated by measuring the Volta-potential and by spectroscopy. Co 3+ - and Fe 3+ -ethioporphyrins were found to be active catalysts, while metal free ethioporphyrins did not display catalytic properties. The oxidation kinetics of substrate in the aqueous phase was studied and the reduced forms of electron acceptors—vitamin K 3 and 1,4-naphthoquinone-were recorded. The interaction of a metal complex of porphyrins with water was also studied.

Properties of hepatic hexose-6-phosphate dehydrogenase purified from brook trout and lake trout

1. Brook trout hexose-6-phosphate dehydrogenase (H6PD) and lake trout H6PD are essentially identical in several parameters including pH optima, kinetic properties and activation energy. They differ in thermal stability. 2. The various oxidations catalyzed by trout H6PD differ in their response to the presence of Mg 2+, ATP and several anions. 3. The apparent K m's of all four substrates show a decrease with decreasing temperature. 4. Kinetics of substrate oxidation by H6PD suggest that the enzyme most likely functions as a G6PD.