scholarly journals Reconstitution of the quinoprotein methanol dehydrogenase from inactive Ca2+-free enzyme with Ca2+, Sr2+ or Ba2+

1996 ◽  
Vol 319 (3) ◽  
pp. 839-842 ◽  
Author(s):  
Matthew G GOODWIN ◽  
Alain AVEZOUX ◽  
Simon L DALES ◽  
Christopher ANTHONY
Keyword(s):  
Activation Energy ◽  
Methanol Dehydrogenase ◽  
Biological Processes ◽  
The Novel ◽  
Methylobacterium Extorquens ◽  
Disulphide Bridge ◽  
Ph Optimum ◽  
Large Conformational Change ◽  
Process Studies ◽  
Time Courses

The reconstitution of active holoenzyme containing calcium from inactive calcium-free methanol dehydrogenase, isolated from a moxA mutant of Methylobacterium extorquens, has a pH optimum of about pH 10, with a well defined pK for the process at pH 9.3. Two Ca2+ ions were irreversibly incorporated per α2β2 tetramer. Calcium could be replaced in the incorporation process by strontium or barium, the affinities for these ions being similar to that for Ca2+. Arrhenius plots for measurement of the activation energy of reconstitution were biphasic; the lower activation energy was typical of most biological processes, while the higher activation energy was at least three times greater, implying the involvement of a large conformational change during incorporation of the cations. The activation energy for incorporation of Ba2+ was considerably higher than that for incorporation of Ca2+. The novel disulphide bridge that is at the active site of the enzyme was not involved in the incorporation process. Studies of the time courses for incorporation of 45Ca2+, production of active enzyme and changes in absorption spectra failed to show any intermediates in the incorporation process.

scholarly journals The role of the novel disulphide ring in the active site of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens

1995 ◽  
Vol 307 (3) ◽  
pp. 735-741 ◽  
Author(s):  
A Avezoux ◽  
M G Goodwin ◽  
C Anthony
Keyword(s):  
Active Site ◽  
Hydrogen Transfer ◽  
Ring Structure ◽  
Methanol Dehydrogenase ◽  
Active Enzyme ◽  
The Novel ◽  
Methylobacterium Extorquens ◽  
Disulphide Bridge ◽  
Prosthetic Group ◽  
Cytochrome Cl

All cysteines in methanol dehydrogenase (MDH) from Methylobacterium extorquens are involved in intra-subunit disulphide bridge formation. One of these is between adjacent cysteine residues which form a novel ring structure in the active site. It is readily reduced, the reduced enzyme being inactive in electron transfer to cytochrome cL. The inactivation is not a result of major structural change or to modification of the prosthetic group pyrrolo-quinoline quinone (PQQ). The reduced enzyme appears to remain active with the artificial electron acceptor phenazine ethosulphate but this is because the dye re-oxidizes the adjacent thiols back to the original disulphide bridge. No free thiols were detected during the reaction cycle with cytochrome cL. Carboxymethylation of the thiols produced by reduction of the novel disulphide ring led to formation of active enzyme. Reconstitution of inactive Ca(2+)-free MDH with Ca2+ led to active enzyme containing the oxidized bridge and reduced quinol, PQQH2, consistent with the conclusion that no hydrogen transfer occurs between these groups in the active site. It is concluded that the disulphide ring in the active site of MDH does not function as a redox component of the reaction. The disulphide ring has no special function in the process of Ca2+ incorporation into the active site. It is suggested that this novel structure might function in the stabilization or protection of the free radical semiquinone form of the prosthetic group (PQQH.) from solvent at the entrance to the active site.

scholarly journals Structure of the quinoprotein glucose dehydrogenase of Escherichia coli modelled on that of methanol dehydrogenase from Methylobacterium extorquens

1995 ◽  
Vol 312 (3) ◽  
pp. 679-685 ◽  
Author(s):  
G E Cozier ◽  
C Anthony
Keyword(s):  
Active Site ◽  
Glucose Dehydrogenase ◽  
Ring Structure ◽  
Enzymic Activity ◽  
Methanol Dehydrogenase ◽  
The Novel ◽  
Methylobacterium Extorquens ◽  
Prosthetic Group ◽  
Site Structure ◽  
Proposed Model

The structure of methanol dehydrogenase (MDH) at 0.194 nm (1.94 A) has been used to provide a model structure for part of a membrane quinoprotein glucose dehydrogenase (GDH). The basic superbarrel structure is retained, along with the tryptophan-docking motifs. The active-site regions are similar, but there are important differences, the most important being that GDH lacks the novel disulphide ring structure formed from adjacent cysteines in MDH; in GDH the equivalent region is occupied by His-262. Because of the overall similarities in the active-site region, the mechanism of action of GDH is likely to be similar to that of MDH. The differences in co-ordination to the cation and bonding to the pyrrolo-quinoline quinone (PQQ) in the active site may explain the relative ease of dissociation of the prosthetic group from the holo-GDH. There are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site, the configuration of which influences substrate specificity. The proposed model is consistent in many respects with previous proposals for the active-site structure based on the effects of chemical modification on binding of PQQ and enzymic activity.

STUDIES ON SOLUBILIZED ADENYLATE KINASE FROM MITOCHONDRIA

1966 ◽  
Vol 44 (11) ◽  
pp. 1469-1475 ◽  
Author(s):  
Marjorie A. Brewster ◽  
Ezzat S. Younathan
Keyword(s):  
Activation Energy ◽  
Rat Liver ◽  
Adenylate Kinase ◽  
Divalent Cations ◽  
Metabolic Function ◽  
Kcal Mole ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Sensitive Method

Adenylate kinase from mitochondria of rat liver was made soluble by sonication. The enzyme had a pH optimum of 8.0, temperature optimum of 30°, and activation energy of 12.2 kcal/mole. It was activated by several divalent cations in the following order of efficiency: Mg++ > Co++ > Mn++ > Ca++, with an optimal Mg++: ADP ratio of 1. The apparent Km value (ADP as substrate) was found to be 1.3 mM at pH 7.4 and 30°. The activity was sensitive to phloretin and mildly activated by aurovertin. Oligomycin, 2,4-dinitrophenol, p-chloromercuribenzoate, alloxan, and phlorizin had no effect on the activity. The metabolic function and a comparison of the properties of this solubilized mitochondrial adenylate kinase with those of similar preparations from other sources are discussed in the light of these findings. During this study, a sensitive method adaptable for a large number of assays of adenylate kinase was developed, and is described in detail.

scholarly journals Ferroptosis-mediated Crosstalk in the Tumor Microenvironment Implicated in Cancer Progression and Therapy

2021 ◽  
Vol 9 ◽  
Author(s):  
Yini Liu ◽  
Chunyan Duan ◽  
Rongyang Dai ◽  
Yi Zeng
Keyword(s):  
Cell Death ◽  
Tumor Microenvironment ◽  
Cancer Progression ◽  
Cancer Biology ◽  
Lipid Peroxides ◽  
Antioxidant Systems ◽  
Biological Processes ◽  
The Novel ◽  
Regulated Cell Death ◽  
Novel Therapeutic Strategies

Ferroptosis is a recently recognized form of non-apoptotic regulated cell death and usually driven by iron-dependent lipid peroxidation and has arisen to play a significant role in cancer biology. Distinct from other types of cell death in morphology, genetics, and biochemistry, ferroptosis is characterized by the accumulation of lipid peroxides and lethal reactive oxygen species controlled by integrated oxidant and antioxidant systems. Increasing evidence indicates that a variety of biological processes, including amino acid, iron, lactate, and lipid metabolism, as well as glutathione, phospholipids, NADPH, and coenzyme Q10 biosynthesis, are closely related to ferroptosis sensitivity. Abnormal ferroptotic response may modulate cancer progression by reprogramming the tumor microenvironment (TME). The TME is widely associated with tumor occurrence because it is the carrier of tumor cells, which interacts with surrounding cells through the circulatory and the lymphatic system, thus influencing the development and progression of cancer. Furthermore, the metabolism processes play roles in maintaining the homeostasis and evolution of the TME. Here, this review focuses on the ferroptosis-mediated crosstalk in the TME, as well as discussing the novel therapeutic strategies for cancer treatment.

scholarly journals Functional investigation of methanol dehydrogenase-like protein XoxF in Methylobacterium extorquens AM1

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2575-2586 ◽  
Author(s):  
Sabrina Schmidt ◽  
Philipp Christen ◽  
Patrick Kiefer ◽  
Julia A. Vorholt
Keyword(s):  
Growth Parameters ◽  
Methanol Dehydrogenase ◽  
Plant Colonization ◽  
Kinetic Properties ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Experimental Conditions ◽  
Dependent Protein ◽  
One Carbon Metabolism ◽  
Functional Investigation

Methanol dehydrogenase-like protein XoxF of Methylobacterium extorquens AM1 exhibits a sequence identity of 50 % to the catalytic subunit MxaF of periplasmic methanol dehydrogenase in the same organism. The latter has been characterized in detail, identified as a pyrroloquinoline quinone (PQQ)-dependent protein, and shown to be essential for growth in the presence of methanol in this methylotrophic model bacterium. In contrast, the function of XoxF in M. extorquens AM1 has not yet been elucidated, and a phenotype remained to be described for a xoxF mutant. Here, we found that a xoxF mutant is less competitive than the wild-type during colonization of the phyllosphere of Arabidopsis thaliana, indicating a function for XoxF during plant colonization. A comparison of the growth parameters of the M. extorquens AM1 xoxF mutant with those of the wild-type during exponential growth revealed a reduced methanol uptake rate and a reduced growth rate for the xoxF mutant of about 30 %. Experiments with cells starved for carbon revealed that methanol oxidation in the xoxF mutant occurs less rapidly compared with the wild-type, especially in the first minutes after methanol addition. A distinct phenotype for the xoxF mutant was also observed when formate and CO2 production were measured after the addition of methanol or formaldehyde to starved cells. The wild-type, but not the xoxF mutant, accumulated formate upon substrate addition and had a 1 h lag in CO2 production under the experimental conditions. Determination of the kinetic properties of the purified enzyme showed a conversion capacity for both formaldehyde and methanol. The results suggest that XoxF is involved in one-carbon metabolism in M. extorquens AM1.

Ambident reactivity in the reaction of phenoxide ion with 2-N-(2′,4′-dinitrophenyl)- and 2-N-(4′-nitrophenyl)-4,6-dinitrobenzotriazole 1-oxides, new superelectrophiles

1988 ◽  
Vol 66 (7) ◽  
pp. 1712-1719 ◽  
Author(s):  
Erwin Buncel ◽  
Julian M. Dust
Keyword(s):  
Activation Energy ◽  
Adduct Formation ◽  
Reaction Pathways ◽  
Resonance Spectroscopy ◽  
The Novel ◽  
Reaction Series ◽  
Bond Scission ◽  
Energy Reaction ◽  
Successive Removal ◽  
Ambident Nucleophile

Reaction of the novel superelectrophiles 2-N-(2′,4′-dinitrophenyl)- and 2-N-(4′-nitrophenyl)-4,6-dinitrobenzotriazole 1-oxides, 3, and 4, possessing two electrophilic centres, with the ambident nucleophile potassium phenoxide in (CD3)2SO was followed by 400 MHz 1H nuclear magnetic resonance spectroscopy. A dichotomy in the reaction pathways has been observed. With MeO−, attack at C-7 leads to reversible adduct formation, while attack at C-1′ results in irreversible N-2: C-1′ bond scission via the metastable C-1′ adduct. In contrast, the reaction of 3 and 4 with PhO− proceeds by a two-pronged attack: formation of C-7 carbon-bonded phenoxide adducts via the ortho and para carbon sites, and oxygen-based cleavage products by attack at the C-1′ position, accompanied by N-2:C-1′ bond scission, in accord with the ambident reactivity of PhO−. Significantly, in this case reaction of both C-7 and C-1′ is effectively irreversible. Moreover, the reaction of phenoxide with either 3 or 4 shows striking differences compared to the reaction of PhO− with 2-N-(picryl)-4,6-dinitrobenzotriazole 1-oxide, 1. Reaction of PhO− with 1 resulted only in O-attack at C-1′ and N-2:C-1′ bond scission; there was no evidence for C-7 adduct formation via O- or C-attack. This marked difference in behaviour can be attributed to the decreased susceptibility to C-1′ attack exhibited by 3 and 4 as compared to 1 and arises from the successive removal of electron-withdrawing nitro groups from the 2-N′-nitroaryl moiety in the series 1 → 3 → 4. The reactions are discussed on the basis of selectivity considerations and an activation energy/reaction coordinate profile comparing the pathways for both C-attack at C-7 and O-attack at C-l′ as electrophilicity (delocalizability) is progressively modulated in the reaction series.

Correlation between effective activation energy and pre-exponential factor for diffusion in bulk metallic glasses

1999 ◽  
Vol 14 (8) ◽  
pp. 3200-3203 ◽  
Author(s):  
S. K. Sharma ◽  
F. Faupel
Keyword(s):  
Activation Energy ◽  
Metallic Glasses ◽  
Effective Activation Energy ◽  
Bulk Metallic Glasses ◽  
Supercooled Liquid ◽  
The Novel ◽  
Effective Activation ◽  
Exponential Factor ◽  
Liquid States ◽  
Fitting Parameters

The values of effective activation energy (Q) and pre-exponential factor (D0) reported in the literature for diffusion in the novel bulk metallic glasses, both in the glassy and the deeply supercooled liquid regions, are found to follow the same correlation as reported earlier in conventional metallic glasses, namely D0 = A exp(Q/B), where A and B are fitting parameters with values A = 4.8 × 10−19 m2 s−1 and B = 0.056 eV atom−1. A possible explanation for the observed values of A and B is given by combining an activation energy and a free volume term. The interpretation favors a cooperative mechanism for diffusion in the glassy and deeply supercooled liquid states.

scholarly journals XoxF Is Required for Expression of Methanol Dehydrogenase in Methylobacterium extorquens AM1

2011 ◽  
Vol 193 (21) ◽  
pp. 6032-6038 ◽  
Author(s):  
E. Skovran ◽  
A. D. Palmer ◽  
A. M. Rountree ◽  
N. M. Good ◽  
M. E. Lidstrom
Keyword(s):  
Methanol Dehydrogenase ◽  
Methylobacterium Extorquens ◽  
Methylobacterium Extorquens Am1

β-Galactosidase from Bacillus stearothermophilus

1976 ◽  
Vol 22 (6) ◽  
pp. 817-825 ◽  
Author(s):  
Richard E. Goodman ◽  
Dennis M. Pederson
Keyword(s):  
Activation Energy ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Bacillus Stearothermophilus ◽  
Thermal Inactivation ◽  
Ph Dependence ◽  
Temperature Sensitive ◽  
Optimum Temperature ◽  
Arrhenius Activation Energy ◽  
Ph Optimum

Several strains of thermophilic aerobic spore-forming bacilli synthesize β-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The β-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 °C and the Arrhenius activation energy is about 24 kcal/mol below 47 °C and 16 kcal/mol above that temperature. At 55 °C the Km is 0.11 M for lactose and 9.8 × 10−3 M for o-nitrophenyl-β-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki = 2.5 × 10−3 M). It is relatively stable at 50 °C, losing only half of its activity after 20 days at this temperature. At 60 °C more than 60% of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein, and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60 °C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000.

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