scholarly journals Dissociation and catalysis in yeast hexokinase A.

1976 ◽  
Vol 155 (3) ◽  
pp. 661-667 ◽  
Author(s):  
D C Williams ◽  
J G Jones
Keyword(s):  
Catalytic Activity ◽  
Steady State ◽  
Ternary Complex ◽  
Specific Activity ◽  
Temperature Dependent ◽  
Gradual Decline ◽  
Experimental Conditions ◽  
Progress Curve ◽  
Catalytically Active ◽  
Yeast Hexokinase

1. The specific activity of yeast hexokinase A depends on the concentration of the protein in the solution being assayed. When a solution containing 13.5 mg of hexokinase A/ml is diluted 10-100-fold at various values of pH and temperature, there is a gradual decline in the specific activity of the enzyme until an equilibrium value is reached, which varies with the chosen experimental conditions. 2. The catalytic activity lost when hexokinase A (1 mg/ml) is incubated at 30degreesC is recovered by lowering the temperature to 25degreesC. 3. These concentration- and temperature-dependent phenomena are consistent with the existence of a monomer-dimer equilibrium in which the dimer alone is the catalytic form of the enzyme. 4. Glucose alone prevents the decline in specific activity of hexokinase A after dilution, but it does not re-activate dilute solutions solutions of the enzyme. It is concluded that glucose binds to both the dimer and the monomer and prevents both association and dissociation. 5. The progress curve describing the phosphorylation of glucose catalysed by hexokinase A does not attain a steady state. It is possible that dissociation of catalytically active dimers in a ternary complex with glucose and ATP (or glucose 6-phosphate and ADP) could explain the non-linearity of this progress curve.

scholarly journals Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

2002 ◽  
Vol 184 (15) ◽  
pp. 4096-4103 ◽  
Author(s):  
Yong Ge ◽  
Frédéric H. Vaillancourt ◽  
Nathalie Y. R. Agar ◽  
Lindsay D. Eltis
Keyword(s):  
Steady State ◽  
Pseudomonas Putida ◽  
Sodium Phosphate ◽  
Ternary Complex ◽  
Specific Activity ◽  
Complex Mechanism ◽  
Aromatic Substrate ◽  
Steady State Kinetics ◽  
Full Complement

ABSTRACT Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe2S2 centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 ± 1 μM, k cat = 3.9 ± 0.2 s−1, and K m O2 = 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°C), where K m O2 represents the K m for O2 and KmA represents the K m for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate ≃ 3-chlorobenzoate > p-toluate ≃ 4-chlorobenzoate ≫ o-toluate ≃ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O2 utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O2 utilization, with >10 times more O2 being consumed than benzoate. However, the apparent K m of TADO for these benzoates was >100 μM, indicating that they do not effectively inhibit the turnover of good substrates.

scholarly journals Steady-state kinetics and inhibition of anaerobically purified human homogentisate 1,2-dioxygenase

2005 ◽  
Vol 386 (2) ◽  
pp. 305-314 ◽  
Author(s):  
Edwin J. A. VELDHUIZEN ◽  
Frédéric H. VAILLANCOURT ◽  
Cheryl J. WHITING ◽  
Marvin M.-Y. HSIAO ◽  
Geneviève GINGRAS ◽  
...  
Keyword(s):  
Steady State ◽  
Active Site ◽  
Ternary Complex ◽  
Specific Activity ◽  
Complex Mechanism ◽  
Substrate Analogues ◽  
Steady State Kinetics ◽  
Km Value ◽  
Substrate Concentrations ◽  
Active Preparation

HGO (homogentisate 1,2-dioxygenase; EC 1.13.11.5) catalyses the O2-dependent cleavage of HGA (homogentisate) to maleylacetoacetate in the catabolism of tyrosine. Anaerobic purification of heterologously expressed Fe(II)-containing human HGO yielded an enzyme preparation with a specific activity of 28.3± 0.6 μmol·min−1·mg−1 (20 mM Mes, 80 mM NaCl, pH 6.2, 25 °C), which is almost twice that of the most active preparation described to date. Moreover, the addition of reducing agents or other additives did not increase the specific activity, in contrast with previous reports. The apparent specificity of HGO for HGA was highest at pH 6.2 and the steady-state cleavage of HGA fit a compulsory-order ternary-complex mechanism (Km value of 28.6±6.2 μM for HGA, Km value of 1240±160 μM for O2). Free HGO was subject to inactivation in the presence of O2 and during the steady-state cleavage of HGA. Both cases involved the oxidation of the active site Fe(II). 3-Cl HGA, a potential inhibitor of HGO, and its isosteric analogue, 3-Me HGO, were synthesized. At saturating substrate concentrations, HGO cleaved 3-Me and 3-Cl HGA 10 and 100 times slower than HGA respectively. The apparent specificity of HGO for HGA was approx. two orders of magnitude higher than for either 3-Me or 3-Cl HGA. Interestingly, 3-Cl HGA inactivated HGO only twice as rapidly as HGA. This contrasts with what has been observed in mechanistically related dioxygenases, which are rapidly inactivated by chlorinated substrate analogues, such as 3-hydroxyanthranilate dioxygenase by 4-Cl 3-hydroxyanthranilate.

scholarly journals Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase

1990 ◽  
Vol 269 (1) ◽  
pp. 261-264 ◽  
Author(s):  
L M A Ferreira ◽  
A J Durrant ◽  
J Hall ◽  
G P Hazlewood ◽  
H J Gilbert
Keyword(s):  
Catalytic Activity ◽  
Specific Activity ◽  
Binding Capacity ◽  
Spatial Separation ◽  
Crystalline Cellulose ◽  
Conserved Sequence ◽  
Binding Domains ◽  
Catalytically Active ◽  
Cellulose Binding ◽  
Derivatives Of

Xylanase A (XYLA) from Pseudomonas fluorescens subspecies cellulosa shows sequence conservation with two endoglucanases from the same organism. The conserved sequence in XYLA, consisting of the N-terminal 234 residues, is not essential for catalytic activity. Full-length XYLA and a fusion enzyme, consisting of the N-terminal 100 residues of XYLA linked to mature alkaline phosphatase, bound tightly to crystalline cellulose (Avicel), but not to xylan. The capacity of truncated derivatives of the xylanase to bind polysaccharides was investigated. XYLA lacking the first 13 N-terminal amino acids did not bind to cellulose. However, a catalytically active XYLA derivative (XYLA′), in which residues 100-234 were deleted, bound tightly to Avicel. Substrate specificity, cellulose-binding capacity, specific activity and Km for xylan hydrolysis were evaluated for each of the xylanases. No differences in any of these parameters were detected for the two enzymes. It is concluded that XYLA contains a cellulose-binding domain consisting of the N-terminal 100 residues which is distinct from the active site. Spatial separation of the catalytic and cellulose-binding domains is not essential for the enzyme to function normally.

scholarly journals The catalytic activity of monomeric yeast hexokinase A

1977 ◽  
Vol 161 (1) ◽  
pp. 181-183
Author(s):  
B P Yip ◽  
F B Rudolph
Keyword(s):  
Catalytic Activity ◽  
Monomeric Form ◽  
Catalytically Active ◽  
Yeast Hexokinase

It has been suggested [Williams, D.C. & Jones, J.G. (1976) Biochem. J. 155, 661-667] that monomeric hexokinase isoenzyme A is not catalytically active. We here present data from reacting-enzyme sedimentation, dissociation experiments and from previous studies which are consistent with the monomeric form possessing catalytic activity.

scholarly journals Fate of the SpoIIAB*-ADP Liberated after SpoIIAB Phosphorylates SpoIIAA of Bacillus subtilis

2000 ◽  
Vol 182 (21) ◽  
pp. 6250-6253 ◽  
Author(s):  
Chung-Sheng Lee ◽  
Isabelle Lucet ◽  
Michael D. Yudkin
Keyword(s):  
Bacillus Subtilis ◽  
Steady State ◽  
Protein Kinase ◽  
Ternary Complex ◽  
Enzymatic Reaction ◽  
Kinetic Studies ◽  
Active State ◽  
Steady State Rate ◽  
Catalytically Active ◽  
State Rate

ABSTRACT Phosphorylation of SpoIIAA catalyzed by SpoIIAB helps to regulate the first sporulation-specific ς factor, ςF, ofBacillus subtilis. The steady-state rate of phosphorylation is known to be exceptionally slow and to be limited by the return of the protein kinase, SpoIIAB, to a catalytically active state. Previous work from this laboratory has suggested that, after catalyzing the phosphorylation, SpoIIAB is in a form (SpoIIAB*) that does not readily release ADP. We now show that the rate of release of ADP from the SpoIIAB*-ADP complex was much diminished by the presence of unreacted SpoIIAA, suggesting that SpoIIAA can form a long-lived ternary complex with SpoIIAB*-ADP in which the SpoIIAB* form is stabilized. In kinetic studies of the phosphorylation of SpoIIAA, the ternary complex SpoIIAA-SpoIIAB*-ADP could be distinguished from the short-lived complex SpoIIAA-SpoIIAB-ADP, which can be readily produced in the absence of an enzymatic reaction.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

Hydrogen peroxide decomposition on a two-component CuO-Cr2O3 catalyst

1988 ◽  
Vol 53 (8) ◽  
pp. 1636-1646 ◽  
Author(s):  
Viliam Múčka ◽  
Kamil Lang
Keyword(s):  
Hydrogen Peroxide ◽  
Catalytic Activity ◽  
Extreme Values ◽  
Catalytic Properties ◽  
Active Components ◽  
Catalytic Systems ◽  
Heterogeneous Catalytic ◽  
Peroxide Decomposition ◽  
Two Component ◽  
Catalytically Active

Some physical and catalytic properties of the two-component copper(II)oxide-chromium(III)oxide catalyst with different content of both components were studied using the decomposition of the aqueous solution of hydrogen peroxide as a testing reaction. It has been found that along to both basic components, the system under study contains also the spinel structure CuCr2O4, chromate washable by water and hexavalent ions of chromium unwashable by water. The soluble chromate is catalytically active. During the first period of the reaction the equilibrium is being established in both homogeneous and heterogeneous catalytic systems. The catalytic activity as well as the specific surface area of the washed solid is a non-monotonous function of its composition. It seems highly probable that the extreme values of both these quantities are not connected with the detected admixtures in the catalytic system. The system under study is very insensitive with regard to the applied doses of gamma radiation. Its catalytic properties are changed rather significantly after the thermal treatment and particularly after the partial reduction to low degree by hydrogen. The observed changes of the catalytic activity of the system under study are very probably in connection with the changes of the valence state of the catalytically active components of the catalyst.

scholarly journals The steady-state visual evoked potential (SSVEP) reflects the activation of cortical object representations: evidence from semantic stimulus repetition

2020 ◽  
Author(s):  
Elise L. Radtke ◽  
Ulla Martens ◽  
Thomas Gruber
Keyword(s):  
Steady State ◽  
Object Representation ◽  
Sensory Information ◽  
Familiar Object ◽  
Object Representations ◽  
Stimulus Repetition ◽  
Experimental Conditions ◽  
Repetition Suppression ◽  
Task Irrelevant ◽  
Visual Evoked

AbstractWe applied high-density EEG to examine steady-state visual evoked potentials (SSVEPs) during a perceptual/semantic stimulus repetition design. SSVEPs are evoked oscillatory cortical responses at the same frequency as visual stimuli flickered at this frequency. In repetition designs, stimuli are presented twice with the repetition being task irrelevant. The cortical processing of the second stimulus is commonly characterized by decreased neuronal activity (repetition suppression). The behavioral consequences of stimulus repetition were examined in a companion reaction time pre-study using the same experimental design as the EEG study. During the first presentation of a stimulus, we confronted participants with drawings of familiar object images or object words, respectively. The second stimulus was either a repetition of the same object image (perceptual repetition; PR) or an image depicting the word presented during the first presentation (semantic repetition; SR)—all flickered at 15 Hz to elicit SSVEPs. The behavioral study revealed priming effects in both experimental conditions (PR and SR). In the EEG, PR was associated with repetition suppression of SSVEP amplitudes at left occipital and repetition enhancement at left temporal electrodes. In contrast, SR was associated with SSVEP suppression at left occipital and central electrodes originating in bilateral postcentral and occipital gyri, right middle frontal and right temporal gyrus. The conclusion of the presented study is twofold. First, SSVEP amplitudes do not only index perceptual aspects of incoming sensory information but also semantic aspects of cortical object representation. Second, our electrophysiological findings can be interpreted as neuronal underpinnings of perceptual and semantic priming.

scholarly journals Role of CO2 During Oxidative Dehydrogenation of Propane Over Bulk and Activated-Carbon Supported Cerium and Vanadium Based Catalysts

2021 ◽  
Author(s):  
Petar Djinović ◽  
Janez Zavašnik ◽  
Janvit Teržan ◽  
Ivan Jerman
Keyword(s):  
Catalytic Activity ◽  
Activated Carbon ◽  
Oxidative Dehydrogenation ◽  
Active Phase ◽  
Lattice Oxygen ◽  
Chemisorbed Oxygen ◽  
Catalytically Active ◽  
Temperature Programmed ◽  
Propane Cracking

AbstractCeO2, V2O5 and CeVO4 were synthesised as bulk oxides, or deposited over activated carbon, characterized by XRD, HRTEM, CO2-TPO, C3H8-TPR, DRIFTS and Raman techniques and tested in propane oxidative dehydrogenation using CO2. Complete oxidation of propane to CO and CO2 is favoured by lattice oxygen of CeO2. The temperature programmed experiments show the ~ 4 nm AC supported CeO2 crystallites become more susceptible to reduction by propane, but less prone to re-oxidation with CO2 compared to bulk CeO2. Catalytic activity of CeVO4/AC catalysts requires a 1–2 nm amorphous CeVO4 layer. During reaction, the amorphous CeVO4 layer crystallises and several atomic layers of carbon cover the CeVO4 surface, resulting in deactivation. During reaction, V2O5 is irreversibly reduced to V2O3. The lattice oxygen in bulk V2O5 favours catalytic activity and propene selectivity. Bulk V2O3 promotes only propane cracking with no propene selectivity. In VOx/AC materials, vanadium carbide is the catalytically active phase. Propane dehydrogenation over VC proceeds via chemisorbed oxygen species originating from the dissociated CO2. Graphic Abstract

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