Showing papers on "Substrate (chemistry) published in 1988"

The capacity of hydrogenotrophic anaerobic bacteria to compete for traces of hydrogen depends on the redox potential of the terminal electron acceptor

Abstract: The effect of different electron acceptors on substrate degradation was studied in pure and mixed cultures of various hydrogenotrophic homoacetogenic, methanogenic, sulfate-reducing, fumarate-reducing and nitrate-ammonifying bacteria. Two different species of these bacteria which during organic substrate degradation produce and consume hydrogen, were cocultured on a substrate which was utilized only by one of them. Hydrogen, which was excreted as intermediate by the first strain (and reoxidized in pure culture), could, depending on the hydrogen acceptor present, also be used by the second organism, resulting in interspecies hydrogen transfer. The efficiency of H2 transfer was similar when methanol, lactate or fructose were used as organic substrate, although the free energy changes of fermentative H2 formation of these substrates are considerably different. In coculture experiments nitrate or fumarate>sulfate> CO2/CH4>sulfur or CO2/acetate were the preferred electron acceptors, and an increasing percentage of H2 was transferred to that bacterium which was able to utilize the preferred electron acceptor. In pure culture the threshold values for hydrogen oxidation decreased in the same order from ≤1,100 ppm for homoacetogenic bacteria to about 0.03 ppm for nitrate or fumarate reducing bacteria. The determined H2-threshold values as well as the percentage of H2 transfer in cocultures were related to the Gibbs free energy change of the respective hydrogen oxidizing reaction.

Manganese peroxidase from the basidiomycete Phanerochaete chrysosporium: spectral characterization of the oxidized states and the catalytic cycle.

Abstract: Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the Mn(II)-dependent oxidation of a variety of phenols. Herein, the authors spectroscopically characterize the oxidized states of MnP compounds I, II, and III and clarify the role of Mn in the catalytic cycle of the enzyme. Addition of 1 equiv of H/sub 2/O/sub 2/ to the native ferric enzyme yields compound I, characterized by absorption maxima at 407, 558, 605, and 650 nm. Addition of 2 or 250 equiv of H/sub 2/O/sub 2/ to the native enzyme yields compound II or III, respectively, identified by absorption maxima at 420, 528, and 555 nm or at 417, 545, and 579 nm, respectively. These characteristics are very similar to those of horseradish peroxidase (HRP) and lignin peroxidase (LiP) compounds I, II, and III. Addition of 1 equiv of either Mn(II), ferrocyanide, or a variety of phenols to MnP compound I rapidly reduces it to MnP compound II. In contrast, only Mn(II) or ferrocyanide, added at a concentration of 1 equiv, reduces compound II. The Mn(III) produced by the enzymic oxidation of Mn(II) oxidizes the terminal phenolic substrates. This indicates that compounds I and II of MnP contain 2more » and 1 oxidizing equiv, respectively, over the native ferric resting enzyme and that the catalytic cycle of the enzyme follows the path native enzyme ..-->.. compound I ..-->.. compound II ..-->.. native enzyme. In addition, these results indicate that Mn(II) serves as an obligatory substrate for MnP compound II, allowing the enzyme to complete its catalytic cycle. Finally, the Mn(II)/Mn(III) redox couple enables the enzyme to rapidly oxidize the terminal phenolic substrates.« less

Two-dimensional diffusion glucose substrate sensing electrode

Abstract: Enzyme electrode assembly suitable for sensing physiologically important molecules such as glucose wherein the rate of accessibility of enzyme substrates to the enzyme is differentially regulated by suitably positioning material with selective permeability properties around the sensing region of the electrode assembly.

Changes in substrate metabolism and effects of excess fatty acids in reperfused myocardium.

Abstract: The purpose of these studies was to characterize the rates of fatty acid oxidation in reperfused myocardium and test the influence of excess fatty acids (FA) on mechanical function in the extracorporeally perfused, working swine heart model. Seventeen animals were prepared. Eight were untreated (LOW FA group; serum FA averaged 0.55 +/- 0.07 mumol/ml) and nine received a constant infusion of 10% Intralipid with heparin to raise serum FA to about 1.4 +/- 0.21 mumol/ml (HIGH FA group). Coronary flow in both groups was held at aerobic levels for an equilibrium period of 40 minutes, acutely reduced regionally in the anterior descending circulation by 60% for 45 minutes, and acutely restored to aerobic levels for 60-minute reflow. Appreciable mechanical depression (-47 delta% from aerobic values; p less than 0.01) during reperfusion was noted in both groups. This was associated with modest reductions in myocardial oxygen consumption (p less than 0.05) and losses of total tissue carnitine stores (p at least less than 0.02). Reperfused myocardium showed a strong preference for and aerobic use of FA during reflow such that 14CO2 production from labeled palmitate exceeded preischemic levels (+89 delta% in LOW FA hearts; +111 delta% in HIGH FA hearts). This suggested relative preservation of restoration of certain elements in mitochondrial function during reflow. The findings argue for uncoupling between substrate metabolism and energy production, accelerated but useless energy drainage, or some impairment between energy transfer and function of contractile proteins as possible explanations for the persistent depression of mechanical function (stunning) during reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbial Decomposition in Aquatic Environments: Combined Process of Extracellular Enzyme Activity and Substrate Uptake

Abstract: The aim of this study was to define a model for the coupling between extracellular enzyme activity and substrate uptake by bacterial populations in natural waters. The balance between uptake of leucine and extracellular hydrolytic production of leucine from a peptide model substrate was investigated in a combined fluorescence-radiotracer experiment with [3H]leucine as a marker for the leucine pool and l-leucine-4 methyl-7-coumarinylamide (Leu-MCA) as a marker for the pool of dissolved peptide substrates. Results show that at low concentrations of the model substrate the input and uptake processes of leucine are nearly balanced, whereas at high concentrations of the model substrate much more leucine is liberated than taken up. In addition, samples from one polluted and one less polluted station in the Kiel Fjord were investigated for their extracellular enzymatic and uptake properties in an annual cycle. It was found that turnover rates of leucine (Tr, percent per hour) and hydrolysis rates of Leu-MCA (Hr, percent per hour), as well as the quotient Tr/Hr, reflect the impact of environmental conditions on decomposition processes at both sampling sites. The quotient Tr/Hr is interpreted as an indirect measurement of the pool size ratio (polymers/monomers), which may serve as an index of hydrolysis-uptake coupling in bacterial utilization of dissolved protein. Calculated on an annual average basis, turnover rates are ca. nine times higher than hydrolysis rates at the polluted station and ca. five times higher at the less polluted station. From the described model, this would mean that the relative fraction of polymers within the total dissolved organic carbon pool (with regard to the substrate combination dissolved protein-leucine) is about twice that at the polluted than at the less polluted station.

Immobilized enzyme electrodes

Abstract: Enzyme electrodes are disclosed which are capable of responding amperometrically to the catalytic activity of the enzyme in the presence of its respective substrate and comprising the enzyme immobilized or adsorbed onto the surface of an electrically conductive support member which consists of or comprises a porous layer of resin-bonded carbon or graphite particles, said particles having intimately mixed therewith, or deposited or adsorbed onto the surface of the individual particles prior to bonding to form said layer, a finely divided platinum group metal, thereby to form a porous, substrate layer onto which said enzyme is adsorbed or immobilized and comprising a substantially heterogeneous layer of resin-bonded carbon or graphite particles, with said platinum group metal dispersed substantially uniformly throughout said layer. The preferred substrate materials are resin bonded platinized carbon paper electrodes comprising platinized carbon powder particles having colloidal platinum adsorbed on the surface of the particles and bonded onto a carbon paper substrate using a synthetic resin, preferably polytetrafluoroethylene, as the binder. The preferred enzyme electrodes are glucose oxidase electrodes comprising glucose oxidase adsorbed or immobilized onto the surface of the substrate.

Triosephosphate isomerase catalysis is diffusion controlled. Appendix: Analysis of triose phosphate equilibria in aqueous solution by 31P NMR.

Abstract: The rates of the forward and reverse reactions of triosephosphate isomerase catalyzed by the wild-type and by a sluggish mutant enzyme have been studied in the absence and the presence of several viscosogenic agents. For the mutant enzyme, the kcat for which is some 10(3) times less than that for the wild-type enzyme, the value of kcat/Km with glyceraldehyde phosphate as substrate is almost unaffected by the presence of sucrose or glycerol, even though the concentration of the aldehyde form of the substrate is smaller because of hemiacetal formation. [The nature and relative amounts of the various forms of triose phosphate present in solution (free carbonyl forms, hydrates, dimers, hemiacetal adducts) have been evaluated by 31P NMR and are presented in the Appendix.] The viscosogenic agents cause the substrate to bind more tightly to the enzyme, roughly compensating for the lower substrate concentration. With dihydroxyacetone phosphate as substrate, the values of kcat/Km for the mutant enzyme increase with the addition of viscosogenic agent, consistent with the tighter binding of substrate without (in this case) any concomitant loss due to hemiketal formation. These results for the mutant enzyme (known to be limited in rate by an enolization step in the catalytic mechanism) can be used to interpret the behavior of the wild-type enzyme. Plots of the relative values of kcat/Km for catalysis by the wild-type enzyme (normalized with the corresponding data for the mutant enzyme) against the relative viscosity have slopes close to unity, as predicted by the Stokes-Einstein equation for a cleanly diffusive process. In the presence of polymeric viscosogenic additives such as poly(ethylene glycol), polyacrylamide, or ficoll, no effect on kcat/Km is seen for the wild-type enzyme, consistent with the expectation that molecular diffusion rates are unaffected by the macroviscosity and are only slowed by the presence of smaller agents that raise the microviscosity. These results show that the reaction catalyzed by the wild-type triosephosphate isomerase is limited by the rate at which glyceraldehyde phosphate encounters, or departs from, the active site.

Mode of activation and kinetic properties of three distinct forms of protein kinase C from rat brain.

Abstract: Three types of protein kinase C, designated types I, II, and III, were purified from rat brain cytosol, and have been shown to correspond to the cDNA clones gamma, beta, and alpha, respectively. Their relative activities in the whole brain tissue were roughly 26, 49, and 25% with H1 histone as a substrate. Type II enzyme was an unequal mixture of two subspecies (roughly 1:7) encoded by beta I and beta II sequences which differ from each other only in a short range of their carboxyl-terminal end regions. Although the three types have closely similar structures, they showed slightly different modes of activation and kinetic properties. Type I enzyme was less sensitive to diacylglycerol but was significantly activated by low concentrations of free arachidonic acid. Type II enzyme exhibited substantial activity without elevated Ca2+ levels, and responded well to diacylglycerol and, to some extent, arachidonic acid. The type III enzyme responded to diacylglycerol as well as to arachidonic acid. The mode of activation of the enzyme by arachidonic acid required elevated levels of Ca2+ but not phospholipid. In the presence of phospholipid, phorbol esters could activate all three types in a manner similar to diacylglycerol. Among various phospholipids tested, phosphatidylserine was the most effective for all three types. Type III enzyme was most sensitive to 1-stearoyl-2-arachidonylglycerol for activation. Conversely, type I enzyme was activated most efficiently by synthetic permeable diacylglycerols, such as 1,2-didecanoylglycerol and 1,2-dioctanoylglycerol. Many heavy metal ions exerted variable and distinct effects on the catalytic activities of these three types.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the protein moiety of ribonuclease P, a ribonucleoprotein enzyme

Abstract: The Bacillus subtilis ribonuclease P consists of a protein and an RNA. At high ionic strength the reaction is protein-independent; the RNA alone is capable of cleaving precursor transfer RNA, but the turnover is slow. Kinetic analyses show that high salt concentrations facilitate substrate binding in the absence of the protein, probably by decreasing the repulsion between the polyanionic enzyme and substrate RNAs, and also slow product release and enzyme turnover. It is proposed that the ribonuclease P protein, which is small and basic, provides a local pool of counter-ions that facilitates substrate binding without interfering with rapid product release.

A model of enzyme adsorption and hydrolysis of microcrystalline cellulose with slow deactivation of the adsorbed enzyme

Abstract: Reduction in the activity and the concentration of the adsorbed enzyme are noted in the experimental data. Two alternative mechanisms, inactivation of the adsorbed enzyme and mass transfer of the enzyme from the bulk solution to the solution within the cellulose fibril where the cellulase is assumed to be inactive, are used to represent the decline in activity. The decline in concentration of the adsorbed enzyme is represented by a modest product inhibition and, more importantly, the assumption that the concentration of the adsorption sites is proportional to the square of the remaining substrate concentration. Measurements of both adsorbed enzyme and product concentration over time are used in determining parameter values. The model is applied to a series of experiments having a 10-fold range of substrate concentration and to an experiment in which the product is removed continuously. For both deactivation mechanisms, a very good representation of product concentration (standard deviation 3.6%) is obtained over the full period (168 h) of hydrolysis; the representation of adsorbed enzyme is, however, less accurate (standard deviation 6.7-6.8%).

Electrochemical cncentration detector method

Abstract: Electrochemical biosensor systems or apparatus, components thereof, and methods for quantitatively measuring or assaying concentrations of selective substrates or metabolites in aqueous media as a function of electrochemical response to hydrogen peroxide concentration, using immobilized enzyme electrode means selective for the substrate or metabolite being measured.

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556.

Abstract: From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45 degrees C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight at D = 0.1 h to 2 U mg of cell dry weight at D = 0.8 h. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.

Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity

Abstract: Chemical synthesis is described of a 77-nucleotide-long RNA molecule that has the sequence of an Escherichia coli Ado-47-containing tRNA(fMet) species in which the modified nucleosides have been substituted by their unmodified parent nucleosides. The sequence was assembled on a solid-phase, controlled-pore glass support in a stepwise manner with an automated DNA synthesizer. The ribonucleotide building blocks used were fully protected 5'-monomethoxytrityl-2'-silyl-3'-N,N-diisopropylaminophosphoram idites. p-Nitro-phenylethyl groups were used to protect the O6 of guanine residues. The fully deprotected tRNA analogue was characterized by polyacrylamide gel electrophoresis (sizing), terminal nucleotide analysis, sequencing, and total enzyme degradation, all of which indicated that the sequence was correct and contained only 3-5 linkages. The 77-mer was then assayed for amino acid acceptor activity by using E. coli methionyl-tRNA synthetase. The results indicated that the synthetic product, lacking modified bases, is a substrate for the enzyme and has an amino acid acceptance 11% of that of the major native species, tRNA(fMet) containing 7-methylguanosine at position 47.

Purification and properties of benzoate-coenzyme A ligase, a Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate.

Abstract: A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudomonas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light with benzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite, phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation with a specific activity of 25 mumol/min per mg of protein and a molecular weight of 60,000. The purified enzyme was insensitive to oxygen and catalyzed the Mg2+ ATP-dependent formation of acyl-CoA from carboxylate and free reduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent Km values of 0.6 to 2 microM for benzoate, 2 to 3 microM for ATP, and 90 to 120 microM for reduced CoA were determined. The reaction product, benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match the kinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry of benzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris. Images

Production of Biodispersan by Acinetobacter calcoaceticus A2.

Abstract: Bacterial strains A2 and HE5, isolated by enrichment culture techniques, were shown to produce extracellular, nondialyzable materials which disperse limestone powders in water. These materials are referred to as biodispersans. Strains A2 and HE5 were classified as Acinetobacter calcoaceticus by physiological and genetic tests. An assay for limestone-dispersing activity was developed which is based on the settling time of a standard 10% limestone-in-water mixture. The assay was proportional to biodispersan concentration between 40 and 100 μg/ml. Dispersion was optimum between pH 9 and 12. Phosphate (2 mM) and magnesium (8 mM) ions caused a 50% inhibition of activity. An optimized medium for the production of biodispersan was developed with ethanol as the substrate. Biodispersan was produced only after the bacteria completed their exponential growth phase and continued during the stationary phase, reaching over 4 g of crude biodispersan per liter. Images

Aspergillus ficuum phytase: partial primary structure, substrate selectivity, and kinetic characterization.

Abstract: Purified Aspergillus ficuum phytase's partial primary structure and amino acid and sugar composition were elucidated. Determination of kinetic parameters of the enzyme at different pH values and temperatures indicated no significant alteration of the Km for phytate while the Kcmt was affected. The enzyme was able to release more than 512 of the total available P1 from phytate in a 3.0 hr assay at 58°C, but the Kcmt dropped to 15Z of the initial rate. Substrate selectivity studies revealed phytate to be the preferred substrate. The pH optima of phytase was 5.0, 4.0, and 3.0 for phytate, ATP, and polyphosphate, respectively. The enzyme had varied sensitivity towards cations. While Ca±± and Fe±±produced no effect on the catalytic rate of the enzyme, Cu±, Cu±±, Zn±±, and Fe±±± were found to be inhibitory. Mn±± was observed to enhance enzyme activity by 33Z at 50 μM. Known inhibitors of acid phosphatases e. g. L (±)-tartrate, phosphomycin, and sodium fluoride had no effect on enzyme activity.

A note on the use of dinitrosalicylic acid for determining the products of enzymatic reactions

Abstract: Factors contributing to non-linearity of enzyme assays incorporating detection of reaction products using dinitrosalicylic acid (DNS) are discussed. The common practice of diluting reaction products before quantification of reducing compounds is shown to be one cause of non-linearity. Insufficiency of substrate is also an important contributory factor in most modern versions of the method, although the original procedure was correctly designed to ensure a linear reaction. The inherent inaccuracy involved in expression of the results of non-linear reactions in units implying linearity (katals or International Units) is emphasized.

Remazol Brilliant Blue-xylan: A soluble chromogenic substrate for xylanases

Abstract: Publisher Summary The conjugate of the polysaccharide with the dye, abbreviated as Remazol Brilliant Blue-Xylan (RBB-xylan), represents a convenient substrate for determination of activity and detection of xylanases. This chapter describes assay method that is based on photometric measurements of the enzyme released dyed fragments soluble in the presence of two volumes of ethanol that precipitates the original substrate and its high-molecular-weight fragments. This principle offers to measure xylanase activity in cell-free extracts and media containing larger amount of reducing compounds that would interfere with classical xylanase activity determination. Xylanase activity on the cell surface and on isolated membranes and organelles can be followed in the presence of viable cells consuming xylose and xylooligosaccharides. The detection of xylanase activity in gels employs transparent agar replicas containing RBB-xylan. Diffusion of dyed fragments released in the place of the enzyme into the separation gel or their selective removal from the agar replicas by the solvent system used for the precipitation of unhydrolyzed RBB-xylan in a solution, represents the basis for enzyme detection. A great advantage of the technique is that the substrate present in a 2% agar gel does not precipitate as it does in a solution, so that the agar replicas remain transparent during and after the destaining of the zones of the enzyme-depolymerized substrate.

Bifunctional chelating agents.

Abstract: The present invention provides bifunctional chelating agents comprising a unique substrate reactive moiety incorporated into a carboxymethyl arm of a polyaminopolycarboxylate chelating framework capable of forming stable complexes with metal ions.

Biosynthesis of Δ-aminolevulinate in greening barley leaves. IX. Structure of the substrate, mode of gabaculine inhibition, and the catalytic mechanism of glutamate 1-semialdehyde aminotransferase

Abstract: Glutamic acid 1-semialdehyde hydrochloride was synthesized and purified. Its prior structural characterization was extended and confirmed by1H NMR spectroscopy and chemical analyses. In aqueous solution at pH 1 to 2 glutamic acid 1-semialdehyde exists in a stable hydrated form, but at pH 8.0 it has a half-life of 3 to 4 min. Spontaneous degradation of the material at pH 8.0 generated some undefined condensation products, but coincidentally a significant amount isomerized to 5-aminolevulinate. At pH 6.8 to 7.0, glutamate 1-semialdehyde is sufficiently stable to permit routine and reproducible assay for glutamate 1-semialdehyde aminotransferase activity. Only about 20% of the enzyme extracted from chloroplasts was sensitive to inactivation by gabaculine with no pretreatment. However, when the enzyme was exposed to 5-aminolevulinate, levulinate or 4,5-dioxovalerate in the absence of glutamate 1-semialdehyde, it was completely inactivated by gabaculine; 4,6-dioxoheptanoate had no effect on the enzyme. These results lead to the hypothesis that the aminotransferase exists in the chloroplast in a complex with pyridoxamine phosphate, which must be converted to the pyridoxal form before it can form a stable adduct with gabaculine. We propose that the enzyme catalyzes the conversion of glutamate 1-semialdehyde to 5-aminolevulinate via 4,5-diaminovalerate.

5'-Nucleotidases in rat heart. Evidence for the occurrence of two soluble enzymes with different substrate specificities.

Abstract: Chromatography of soluble proteins from rat heart on phosphocellulose columns separates two 5'-nucleotidases. The first to emerge from the column shows a preference for AMP over IMP as substrate, whereas the second shows a preference for IMP over AMP. The properties of the IMP-preferring enzyme, including the conditions under which it is eluted from phosphocellulose columns, show it to be the enzyme studied by Itoh, Oka & Ozasa [Biochem. J. (1986) 235, 847-851]. The kinetic properties of the AMP-preferring enzyme indicate that it is likely to be the enzyme responsible for the production of adenosine under conditions of hypoxia and increased work load, and with metabolic stresses such as a high load of acetate.

The stromacentre inAvena plastids: an aggregation ofβ-glucosidase responsible for the activation of oat-leaf saponins.

Abstract: The stromacentre, a particular structure in the plastids of mostAvena species, was isolated from etioplasts ofAvena sativa and then characterized to determine its biological function. When comparing differentAvena species with or without stromacentre, it was shown that the stromacentre, a 63-kDa protein, and saponins (characteristic compounds ofAvena sativa) either occur together or not at all. This linkage was confirmed by demonstrating a transformation of saponins by the isolated stromacentre protein: avenacosides were hydrolyzed to 26-desgluco-avenacosides. Therefore, the stromacentre protein had to be regarded as aβ-glucosidase. Enzyme assays usingp-nitrophenyl-β-d-glucopyranoside as substrate showed that thisβ-glucosidase has a pH optimum at pH 6.0. The calculatedK m value for this substrate was 2.2·10-3 M. Antibodies against the stromacentre protein inhibitedβ-glucosidase activity. The determination of the molecular weight of theβ-glucosidase by sodium dodecyl sulfate-gel electrophoresis showed that it consists of subunits of 63 kDa. After gel electrophoresis under non-denaturing conditions, enzymatically active molecules were shown to consist of at least two of these subunits. Molecules aggregated up to about 106 Da also had enzyme activity. Enzyme assays using avenacosides as substrate showed a pH optimum at pH 6.0. The calculatedK m value for this substrate was 1.2·10-5 M. The high affinity to the avenacosides and the high specificity for the C-26 bound glucose indicate that avenacosides are the natural substrates for thisβ-glucosidase. Assuming that the avenacosides in oat leaves play a role as preformed chemical inhibitory substances against phytopathogenic microorganisms, a model is presented showing the stromacentre with a central role in activating the fungitoxicity of avenacosides.

Kinetic characterization of the carbon monoxide-acetyl-CoA (carbonyl group) exchange activity of the acetyl-CoA synthesizing CO dehydrogenase from Clostridium thermoaceticum

Abstract: CO dehydrogenase from Clostridium thermoaceticum is a nickel-containing enzyme that catalyzes both the reversible conversion of CO2 to CO (for incorporation into the carbonyl group of acetate) and the synthesis of acetyl-CoA from methyl corrinoid, CO, and CoASH. The latter activity is conveniently assayed by monitoring the exchange of [1-14C]acetyl-CoA (carbonyl group) with 12CO. Kinetic parameters for the highly oxygen sensitive exchange activity have been determined: Km (acetyl-CoA) = 600 microM; Vmax = 440 min-1. In addition, coenzyme A analogues have been tested as inhibitors of the exchange to probe the active site of the enzyme; each has no effect on the CO2 in equilibrium CO activity of CO dehydrogenase. Coenzyme A, the substrate for acetate biosynthesis, is a potent competitive inhibitor, KI = 7 microM. Comparison of this value with that for desulfo-CoA (KI = 6000 microM) suggests that a key mode of binding is through the sulfur atom, possibly to a metal site on the enzyme. The relatively high affinity of the enzyme for CoASH relative to acetyl-CoA is consistent with its proposed operation in the acetogenic direction. The differential sensitivity to oxygen and storage of the two activities of CO dehydrogenase as well as the contrasting effect of coenzyme A inhibitors suggests that acetate assemblage occurs at a site distinct from that for CO dehydrogenation.