scholarly journals 3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Properties of its acetyl derivative

1985 ◽  
Vol 227 (2) ◽  
pp. 601-607 ◽  
Author(s):  
D M Lowe ◽  
P K Tubbs
Keyword(s):  
Reaction Mechanism ◽  
Half Life ◽  
Thiol Group ◽  
Acetyl Derivative ◽  
Cysteine Residue ◽  
Temperature Dependent ◽  
Acetyl Coa ◽  
Maximum Value ◽  
Affinity Labelling ◽  
Acetyl Group

Ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) reacts with acetyl-CoA to form a complex in which the acetyl group is covalently bound to the enzyme. This acetyl group can be removed by addition of acetoacetyl-CoA or CoA. The extent of acetylation and release of CoA were found to be highly temperature-dependent. At temperatures above 20 degrees C, a maximum value of 0.85 mol of acetyl group bound/mol of enzyme dimer was observed. Below this temperature the extent of rapid acetylation was significantly lowered. Binding stoichiometries close to 1 mol/mol of enzyme dimer were also observed when the 3-hydroxy-3-methylglutaryl-CoA synthase activity was titrated with methyl methanethiosulphonate or bromoacetyl-CoA. This is taken as evidence for a ‘half-of-the-sites’ reaction mechanism for the formation of 3-hydroxy-3-methylglutaryl-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase. The Keq. for the acetylation was about 10. Isolated acetyl-enzyme is stable for many hours at 0 degrees C and pH 7, but is hydrolysed at 30 degrees C with a half-life of 7 min. This hydrolysis is stimulated by acetyl-CoA and slightly by succinyl-CoA, but not by desulpho-CoA. The site of acetylation has been identified as the thiol group of a reactive cysteine residue by affinity-labelling with the substrate analogue bromo[1-14C]acetyl-CoA.

scholarly journals Production and comparison of mature single-domain ‘trefoil’ peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58

1995 ◽  
Vol 308 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
M P Chadwick ◽  
F E B May ◽  
B R Westley
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Gel Filtration ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Exchange Chromatography ◽  
Single Domain ◽  
Trefoil Peptides ◽  
Trefoil Peptide

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the ‘trefoil’ family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.

scholarly journals The extended reductive acetyl-CoA pathway: ATPases in metal cluster maturation and reductive activation

2014 ◽  
Vol 395 (5) ◽  
pp. 545-558 ◽  
Author(s):  
Jae-Hun Jeoung ◽  
Sebastian Goetzl ◽  
Sandra Elisabeth Hennig ◽  
Jochen Fesseler ◽  
Christina Wörmann ◽  
...  
Keyword(s):  
Current Knowledge ◽  
Metal Cluster ◽  
Potential Gradient ◽  
Reductive Activation ◽  
Acetyl Coa ◽  
Oxygen Sensitive ◽  
Metal Organic ◽  
Acetyl Group ◽  
Acetyl Coa Pathway ◽  
Atp Binding And Hydrolysis

Abstract The reductive acetyl-coenzyme A (acetyl-CoA) pathway, also known as the Wood-Ljungdahl pathway, allows reduction and condensation of two molecules of carbon dioxide (CO2) to build the acetyl-group of acetyl-CoA. Productive utilization of CO2 relies on a set of oxygen sensitive metalloenzymes exploiting the metal organic chemistry of nickel and cobalt to synthesize acetyl-CoA from activated one-carbon compounds. In addition to the central catalysts, CO dehydrogenase and acetyl-CoA synthase, ATPases are needed in the pathway. This allows the coupling of ATP binding and hydrolysis to electron transfer against a redox potential gradient and metal incorporation to (re)activate one of the central players of the pathway. This review gives an overview about our current knowledge on how these ATPases achieve their tasks of maturation and reductive activation.

scholarly journals p-NN′-phenylenebismaleimide, a specific cross-linking agent for F-actin

1978 ◽  
Vol 175 (3) ◽  
pp. 1023-1032 ◽  
Author(s):  
P Knight ◽  
G Offer
Keyword(s):  
Quantitative Analysis ◽  
Sodium Dodecyl Sulphate ◽  
Actin Filaments ◽  
Sodium Dodecyl ◽  
Cysteine Residue ◽  
Cross Linking ◽  
Maximum Value ◽  
Cross Links ◽  
The Cross ◽  
Hydrolysis Of

Covalent cross-links can be inserted between the subunits of F-actin by using p-NN′-phenylenebismaleimide. Cross-linking reaches its maximum value when one molecule of reagent has reacted with each actin subunit. p-NN′-Phenylenebismaleimide reacts initially with a cysteine residue on one subunit, the slower cross-linking reaction involving a lysine residue on a neighbouring subunit. Hydrolysis of the actin-bound reagent limits the extent of cross-linking. Quantitative analysis of the amounts of cross-linked oligomers seen on polyacrylamide gels containing sodium dodecyl sulphate suggests that neither the binding of the reagent to actin nor the formation of cross-links introduces strain into the structure. The cross-links do not join together different F-actin filaments, and evidence is presented that suggests that the cross-links join subunits of the same long-pitched helix.

scholarly journals Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus

1976 ◽  
Vol 155 (3) ◽  
pp. 523-534 ◽  
Author(s):  
G Allen ◽  
J I Harris
Keyword(s):  
Conformational Changes ◽  
Bacillus Stearothermophilus ◽  
Three Dimensional ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Three Dimensional Model ◽  
Muscle Enzyme ◽  
Tyrosine Residues ◽  
Pig Muscle

The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.

scholarly journals Oligolysine-based saccharide clusters: synthesis and specificity

2002 ◽  
Vol 368 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Natacha FRISON ◽  
Philippe MARCEAU ◽  
Annie-Claude ROCHE ◽  
Michel MONSIGNY ◽  
Roger MAYER
Keyword(s):  
Plasma Membrane ◽  
Hepatoma Cell ◽  
Surface Membrane ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Cell Uptake ◽  
Amide Bond ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell ◽  
Plant Lectins

In search of specific and highly selective sugar clusters for cell receptors, such as membrane lectins, various disaccharides were coupled to small peptide cores through an amide bond. In a first step, the reducing disaccharides, i.e. lactose and three different dimannoses, were converted into glycosyl-pyroglutamyl-β-alanine derivatives. The free carboxylic group of these conjugates was then coupled to the α and ∊ amino groups of the core peptide (Lysn-Ala-Cys-NH2) with n = 1 to 5, with complete substitution leading to homogeneous glycoclusters. The thiol group of the cysteine residue was used to tag the glycosylated oligolysines upon reaction with fluorescein iodoacetamide. The affinity of these glycoclusters towards two plant lectins was assessed by surface plasmon resonance. The selectivity of their cell uptake was investigated by flow cytometry using two types of cells: a human hepatoma cell line (HepG2 cells) expressing the plasma membrane galactose-specific lectin, and monocyte-derived dendritic cells expressing the plasma membrane mannose-specific lectin. The glycoclusters containing four or five disaccharides were shown to bind plant lectins and cell surface membrane lectins with a narrow selectivity and with a high affinity.

THE SILVER CATALYZED OXIDATION OF ETHYLENE: IV. REACTION MECHANISM

1954 ◽  
Vol 32 (4) ◽  
pp. 443-451 ◽  
Author(s):  
A. Orzechowski ◽  
K. E. MacCormack
Keyword(s):  
Reaction Mechanism ◽  
Ethylene Oxide ◽  
Temperature Dependent ◽  
Heterogeneous Oxidation ◽  
Initial Oxidation ◽  
Partial Pressures ◽  
Parallel Reactions ◽  
Single Oxygen ◽  
Catalyzed Oxidation ◽  
Adsorbed Ethylene

A reaction mechanism for the silver catalyzed oxidation of C2H4 by oxygen has been formulated which is consistent with kinetic data for this system. It is suggested that both ethylene oxide and CO2 formation involve interaction of single gaseous C2H4 molecules with single oxygen adatoms. This may be a system of two parallel reactions of different activation energy requirements or a common initiation step to form adsorbed ethylene oxide which may then desorb immediately or isomerize to acetaldehyde followed by rapid oxidation to CO2 and H2O. Account is taken of the known adsorption characteristics of O2 on silver to deduce expressions for initial rates of ethylene oxide and CO2 formation as a function of reactant partial pressures. The generalized form of the equation is r = k(1 + a/pE + b/pO)−1, where k, a, and b are temperature dependent constants and pE and pO are partial pressures of ethylene and of oxygen respectively.A mechanism is also suggested for the heterogeneous oxidation of ethylene oxide which involves interaction between a gas phase ethylene oxide molecule and a single oxygen adatom to form an intermediate (probably formaldehyde) which is rapidly oxidized to CO2 and H2O. A similar expression to that above for the initial oxidation rate is deduced. These expressions have been fitted successfully to experimental data.

scholarly journals Bicarbonate-dependent ATP cleavage catalysed by pyruvate carboxylase in the absence of pyruvate

1992 ◽  
Vol 287 (3) ◽  
pp. 1011-1017 ◽  
Author(s):  
P V Attwood ◽  
B D L A Graneri
Keyword(s):  
Reaction Mechanism ◽  
Atpase Activity ◽  
Active Site ◽  
Pyruvate Carboxylase ◽  
Substrate Inhibition ◽  
Cleavage Reaction ◽  
Acetyl Coa ◽  
Enzyme Preparations ◽  
Pyruvate Carboxylation ◽  
Rate Of Movement

Preparations of pyruvate carboxylase catalyse the cleavage of MgATP in the absence of pyruvate and acetyl-CoA. The rate of this cleavage is higher in the presence of HCO3- than in its absence. Incubation of the enzyme preparations with an excess of the pyruvate carboxylase inhibitor, avidin, completely abolishes the pyruvate carboxylating activity of the enzyme preparations but only abolishes the HCO3(-)-dependent MgATP cleaving activity, with no effect on the HCO3(-)-independent ATPase activity. The HCO3(-)-dependent MgATP cleavage is also sensitive to inhibition by a pyruvate carboxylase inhibitor, oxamate, and the dependence of the reaction on the free Mg2+ concentration is similar to that of the pyruvate-carboxylation reaction, whereas the HCO3(-)-independent MgATP cleavage is not dependent on the concentration of free Mg2+ in the range tested. This indicates that MgATP cleavage by pyruvate carboxylase is entirely dependent on the presence of HCO3- and that there may be a low level of ATPase contamination in the enzyme preparations. In addition, inhibition of the HCO3(-)-dependent MgATP cleavage by both avidin and oxamate indicate that although biotin does not directly participate in the reaction, its presence is required in that part of the active site of the enzyme. The rate of HCO3(-)-dependent MgATP cleavage is about 0.07% of that of the full pyruvate carboxylation reaction under similar conditions with saturating substrates. The reaction mechanism is sequential with respect to MgATP and HCO3- addition and Mg2+ adds at equilibrium before MgATP. Acetyl-CoA stimulates the HCO3(-)-dependent MgATP cleavage at low MgATP concentrations, with the stimulation being greater at low Mg2+ concentrations. At high levels of MgATP in the presence of acetyl-CoA, substrate inhibition is evident and is more pronounced at increasing concentrations of Mg2+. This inhibition appears to be, at least in part, caused by inhibition of decarboxylation of the enzyme-carboxybiotin complex by the binding to this complex of Mg2+ and MgATP, which probably act to reduce the rate of movement of carboxybiotin from the site of the MgATP cleavage reaction to that of the pyruvate carboxylation reaction where it is unstable and decarboxylates.

scholarly journals Systematic Analysis of a Conserved Region of the Aminoglycoside 6′-N-Acetyltransferase Type Ib

2001 ◽  
Vol 45 (12) ◽  
pp. 3287-3292 ◽  
Author(s):  
Ali Shmara ◽  
Natalia Weinsetel ◽  
Ken J. Dery ◽  
Ramona Chavideh ◽  
Marcelo E. Tolmasky
Keyword(s):  
Amino Acids ◽  
Active Form ◽  
Major Change ◽  
Cysteine Residue ◽  
Alanine Scanning Mutagenesis ◽  
Systematic Analysis ◽  
C Terminus ◽  
Acetyl Group ◽  
Semisynthetic Derivative ◽  
High Level

ABSTRACT Alanine-scanning mutagenesis was applied to the aminoglycoside 6′-N-acetyltransferase type Ib conserved motif B, and the effects of the substitutions were analyzed by measuring the MICs of kanamycin (KAN) and its semisynthetic derivative, amikacin (AMK). Several substitutions resulted in no major change in MICs. E167A and F171A resulted in derivatives that lost the ability to confer resistance to KAN and AMK. P155A, P157A, N159A, L160A, I163A, K168A, and G170A conferred intermediate levels of resistance. Y166A resulted in an enzyme derivative with a modified specificity; it conferred a high level of resistance to KAN but lost the ability to confer resistance to AMK. Although not as pronounced, the resistance profiles conferred by substitutions N159A and G170A were related to that conferred by Y166A. These phenotypes, taken together with previous results indicating that mutant F171L could not catalyze acetylation of AMK when the assays were carried out at 42°C (D. Panaite and M. Tolmasky, Plasmid 39:123–133, 1998), suggest that some motif B amino acids play a direct or indirect role in acceptor substrate specificity. MICs of AMK and KAN for cells harboring the substitution C165A were high, suggesting that the active form of the enzyme may not be a dimer formed through a disulfide bond. Furthermore, this result indicated that the acetylation reaction occurs through a direct mechanism rather than a ping-pong mechanism that includes a transient transfer of the acetyl group to a cysteine residue. Deletion of fragments at the C terminus demonstrated that up to 10 amino acids could be deleted without a loss of activity.

scholarly journals Structural and functional effects of selective chemical modifications of Scapharca inaequivalvis haemoglobins in relation to their unique assembly

1987 ◽  
Vol 241 (2) ◽  
pp. 499-504 ◽  
Author(s):  
A Boffi ◽  
M Gattoni ◽  
R Santucci ◽  
P Vecchini ◽  
F Ascoli ◽  
...  
Keyword(s):  
Functional Properties ◽  
Oxygen Affinity ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Oxygen Binding ◽  
Thiol Groups ◽  
Functional Roles ◽  
Dimeric Unit ◽  
Structural And Functional Properties ◽  
Thiol Reagent

The structural and functional roles of lysyl and thiol groups in the dimeric (HbI) and tetrameric (HbII) haemoglobins from the mollusc Scapharca inaequivalvis have been assessed. In these haemoglobins a unique mode of assembly (the haem-carrying E and F helices form the intersubunit contact of the dimeric unit) is associated with co-operative oxygen binding. Extensive acylation is accompanied by significant haem oxidation. Modification of one or two lysyl residues per chain (corresponding to approximately 20% of the total residues) does not affect the structural and functional properties of both haemoglobins, in line with the proposal that the intersubunit contacts are rich in hydrophobic residues. The modification of the thiol groups does not influence the state of association in both HbI and HbII, despite the location of the cysteine residue common to all polypeptide chains in the vicinity of the major intersubunit contact. The effect on the functional properties depends on the size of the thiol reagent: p-chloromercuribenzoate and phenylmercuric acetate increase the oxygen affinity about 20-fold, but iodoacetamide and mercuric chloride have no effect. Moreover, electrophoresis experiments indicate that p-chloromercuribenzoate is bound in a co-operative fashion, the degree of co-operativity being much higher in the dimeric HbI. Thus, only in HbII are intermediates containing substoichiometric amounts of p-chloromercuribenzoate formed in significant amounts. Their oxygen binding properties show that reaction of only one thiol group/tetramer suffices to alter the oxygen affinity of the molecule.

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