KINETIC MODELS ON ACETYLCHOLINESTERASE MODULATION BY SELF-SUBSTRATE AND POLYAMINES: ESTIMATION OF INTERACTION PARAMETERS AND RATE CONSTANTS FOR FREE AND ACETYLATED STATES OF THE ENZYME

2002 ◽  
Vol 10 (02) ◽  
pp. 127-147 ◽  
Author(s):  
ANDRÉS VENTURINO ◽  
ROSA MARÍA BERGOC ◽  
ANA MARÍA PECHEN DE D'ANGELO ◽  
ENRIQUE ARTURO ROSENBAUM
Keyword(s):  
Substrate Inhibition ◽  
Binding Constants ◽  
Acetylcholinesterase Activity ◽  
Affinity Constant ◽  
Amino Groups ◽  
Activation Free Energy ◽  
Interaction Factor ◽  
Quaternary Complex ◽  
Peripheral Site ◽  
Free Interaction

Polyamines act as dual modulators on electric eel acetylcholinesterase, modifying both the apparent Km and Ki, depending on substrate levels. A kinetic model was developed to explain the results, based on two-step catalysis, a peripheral site for substrate inhibition apart from the catalytic site, and one binding site for polyamine. This model presented the best fittings to data, when compared with a simpler one considering one catalytic step. A fitting equation built up with sixteen independent parameters let us calculate the kinetic constants. In this way, we were able to solve the parameter identifiability problem arising from model uncertainty when only substrate was used in acetylcholinesterase kinetics. Besides, fitting parameters directly provide information about the binding constants of the different complexes, the modulatory strength of substrate and polyamines, and the effect on the standard activation free energy for acetylcholinesterase. Substrate inhibition operates mainly on the first catalytic step with an affinity constant of 5.2 mM-1, which is reduced to one third for the acetylated enzyme. The interaction factor between substrate binding at both sites is about 12. The modulatory strength of polyamines is spermine > spermidine > putrescine. This order is directly related to the number of amino groups in the molecule, and to the calculated free interaction energy. The effect of the number of amino groups on the binding energy is significantly increased in acetylated acetylcholinesterase. It is also inferred that the formation of a quaternary complex enzyme-substrate-substrate-polyamine would not be possible. Some relations between polyamine structure and acetylcholinesterase activity are suggested from estimated constants. Due to the distal amino group distances, it is possible for spermine and spermidine to span along the catalytic gorge of acetylcholinesterase, binding to the catalytic and peripheral sites in a way similar to bisquaternary ammonium inhibitors.

scholarly journals Harnessing Nature’s Diversity: Discovering organophosphate bioscavenger characteristics among low molecular weight proteins

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Reed B. Jacob ◽  
Kenan C. Michaels ◽  
Cathy J. Anderson ◽  
James M. Fay ◽  
Nikolay V. Dokholyan
Keyword(s):  
Binding Constants ◽  
Synaptic Cleft ◽  
Acetylcholinesterase Activity ◽  
Organophosphate Poisoning ◽  
Serine Hydrolase ◽  
Agricultural Pesticides ◽  
Protein Universe ◽  
Hydrolysis Of ◽  
Low Molecular Weight Proteins ◽  
Computational Strategy

Abstract Organophosphate poisoning can occur from exposure to agricultural pesticides or chemical weapons. This exposure inhibits acetylcholinesterase resulting in increased acetylcholine levels within the synaptic cleft causing loss of muscle control, seizures, and death. Mitigating the effects of organophosphates in our bodies is critical and yet an unsolved challenge. Here, we present a computational strategy that integrates structure mining and modeling approaches, using which we identify novel candidates capable of interacting with a serine hydrolase probe (with equilibrium binding constants ranging from 4 to 120 μM). One candidate Smu. 1393c catalyzes the hydrolysis of the organophosphate omethoate (kcat/Km of (2.0 ± 1.3) × 10−1 M−1s−1) and paraoxon (kcat/Km of (4.6 ± 0.8) × 103 M−1s−1), V- and G-agent analogs respectively. In addition, Smu. 1393c protects acetylcholinesterase activity from being inhibited by two organophosphate simulants. We demonstrate that the utilized approach is an efficient and highly-extendable framework for the development of prophylactic therapeutics against organophosphate poisoning and other important targets. Our findings further suggest currently unknown molecular evolutionary rules governing natural diversity of the protein universe, which make it capable of recognizing previously unseen ligands.

Age-related changes in pulmonary muscarinic receptor binding properties

1993 ◽  
Vol 265 (2) ◽  
pp. L103-L109 ◽  
Author(s):  
M. Wills-Karp
Keyword(s):  
Muscarinic Receptor ◽  
Binding Constants ◽  
Affinity Constant ◽  
Receptor Subtypes ◽  
Agonist Binding ◽  
Binding Properties ◽  
Age Related ◽  
Muscarinic Stimulation ◽  
Competition Binding ◽  
Binding Curves

The goal of this study was to elucidate mechanisms responsible for age-related reductions in responsiveness to cholinergic muscarinic stimulation in guinea pigs, by examining the binding properties of muscarinic receptors and their coupling to guanine nucleotide regulatory proteins as a function of animal age. In addition, the binding constants of three selective muscarinic receptor antagonists pirenzepine, [11-((2-[(diethylamino)methyl]-1-piperidinyl)-acetyl)-5, 11-dihydro-6H-pyrido(2,3)(1,4)benzodiazepine-6-on], and 4-diphenylacetoxy-N-methylpiperidine methobromide were examined. We found that there were no changes in either the receptor density or the affinity of the muscarinic receptor with age. There was a significant reduction in the affinity constant for the high-affinity agonist binding site in the old tissues (7.63 +/- 0.08) compared with the young tissues (8.31 +/- 0.10). Guanine nucleotides lowered agonist affinity for the receptor in young lungs, however, they had no effect on agonist binding in old tissues. Antagonist competition binding curves in young tissues revealed that 73% of the receptors are of the M2 type, with 27% being of the M3 subtype. In contrast, antagonist competition binding curves in the old tissues revealed that 37% of the receptors were of the M2 subtype, 30% were M3, and 33% were of the M1 subtype. Our studies provide evidence that the loss of sensitivity to cholinergic muscarinic stimulation in the senescent lung may be due to changes in both muscarinic receptor subtypes and receptor coupling to G proteins.

scholarly journals Effect of Pollyallylamine on Alcoholdehydrogenase Structure and Activity

Polymers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 832
Author(s):  
Aleksandr L. Kim ◽  
Egor V. Musin ◽  
Alexey V. Dubrovskii ◽  
Sergey A. Tikhonenko
Keyword(s):  
Alcohol Dehydrogenase ◽  
Quaternary Structure ◽  
Inhibitory Effect ◽  
Affinity Constant ◽  
Structure And Properties ◽  
Amino Groups ◽  
Sulfate Anion ◽  
Polyelectrolyte Microcapsules ◽  
Adh Activity ◽  
Structure And Activity

In this article, the effect of polyallylamine (PAA) on the structure and catalytic characteristics of alcohol dehydrogenase (ADH) was studied. For this research, we used methods of stationary kinetics and fluorescence spectroscopy. It has been shown that PAA non-competitively inhibits ADH activity while preserving its quaternary structure. It was established that 0.1 M ammonium sulfate removes the inhibitory effect of PAA on ADH, which is explained by the binding of sulfate anion (NH4)2SO4 with polyallylamine amino groups. As a result, the rigidity of the polymer chain increases and the ability to bind to the active loop of the enzyme increases. It is also shown that sodium chloride removes the inhibitory effect of PAA on ADH due to an electrostatic screening of the enzyme from polyelectrolyte. The method of encapsulating ADH in polyelectrolyte microcapsules was adapted to the structure and properties of the enzyme molecule. It was found that the best for ADH is its encapsulation by adsorption into microcapsules already formed on CaCO3 particles. It was shown that the affinity constant of encapsulated alcohol dehydrogenase to the substrate is 1.7 times lower than that of the native enzyme. When studying the affinity constant of ADH in a complex with PAA to ethanol, the effect of noncompetitive inhibition of the enzyme by polyelectrolyte was observed.

scholarly journals Mutation His322Asn in human acetylcholinesterase does not alter electrophoretic and catalytic properties of the erythrocyte enzyme

Blood ◽  
1994 ◽  
Vol 83 (10) ◽  
pp. 3003-3005 ◽  
Author(s):  
P Masson ◽  
MT Froment ◽  
RC Sorenson ◽  
CF Bartels ◽  
O Lockridge
Keyword(s):  
Genetic Variant ◽  
Catalytic Properties ◽  
Substrate Inhibition ◽  
Blood Group Antigen ◽  
Kinetic Properties ◽  
Wild Type ◽  
Exon 2 ◽  
Erythrocyte Enzyme ◽  
Alternatively Spliced ◽  
Peripheral Site

Abstract The YT blood group antigen is located on human red blood cell (RBC) acetylcholinesterase. Wild-type acetylcholinesterase, YT1, has histidine at codon 322, whereas the genetic variant of acetylcholinesterase, YT2, has asparagine. This mutation is located within exon 2 of the ACHE gene, an exon that is present in all alternatively spliced forms of acetylcholinesterase. Therefore, acetylcholinesterase in brain and muscle has the same mutation as RBC acetylcholinesterase. We compared the electrophoretic and kinetic properties of RBC acetylcholinesterases having His 322 or Asn 322. We found no differences in the isoelectric point, mobility on non- denaturing gel electrophoresis, affinity for acetylthiocholine, activity per milligram of RBC ghost protein, substrate inhibition constants, binding to the peripheral site ligand (propidium), and binding to active site ligands (tetrahydroaminoacridine and edrophonium). Thus, although the point mutation elicits antibody production in nonmatching blood transfusion recipients, it has no effect on the enzymatic properties of acetylcholinesterase.

Characterization of Acetylcholinesterase in Caco-2 Cells

2002 ◽  
Vol 227 (7) ◽  
pp. 480-486 ◽  
Author(s):  
Lauren R. Plageman ◽  
Giovanni M. Pauletti ◽  
Kenneth A. Skau
Keyword(s):  
Active Site ◽  
Substrate Inhibition ◽  
Extracellular Fluid ◽  
Acetylcholinesterase Activity ◽  
Monomeric Form ◽  
Catalytic Center ◽  
Membrane Bound ◽  
Degree Of Differentiation ◽  
Triton X

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-β-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.

Substrate Binding to the Peripheral Site of Acetylcholinesterase Initiates Enzymatic Catalysis. Substrate Inhibition Arises as a Secondary Effect†

Biochemistry ◽  
1999 ◽  
Vol 38 (1) ◽  
pp. 122-133 ◽  
Author(s):  
Tivadar Szegletes ◽  
William D. Mallender ◽  
Patrick J. Thomas ◽  
Terrone L. Rosenberry
Keyword(s):  
Substrate Binding ◽  
Substrate Inhibition ◽  
Enzymatic Catalysis ◽  
Secondary Effect ◽  
Peripheral Site

Aldehyde gold clusters for molecular labeling

1995 ◽  
Vol 53 ◽  
pp. 858-859
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya
Keyword(s):  
Covalent Bond ◽  
Aldehyde Group ◽  
Gold Clusters ◽  
Side Reactions ◽  
Amino Groups ◽  
Sodium Cyanoborohydride ◽  
Schiff’S Base ◽  
Protein Molecules ◽  
Hydroxysuccinimide Esters ◽  
Primary Amino

Glutaraldehyde is a useful tissue and molecular fixing reagents. The aldehyde moiety reacts mainly with primary amino groups to form a Schiff's base, which is reversible but reasonably stable at pH 7; a stable covalent bond may be formed by reduction with, e.g., sodium cyanoborohydride (Fig. 1). The bifunctional glutaraldehyde, (CHO-(CH2)3-CHO), successfully stabilizes protein molecules due to generally plentiful amines on their surface; bovine serum albumin has 60; 59 lysines + 1 α-amino. With some enzymes, catalytic activity after fixing is preserved; with respect to antigens, glutaraldehyde treatment can compromise their recognition by antibodies in some cases. Complicating the chemistry somewhat are the reported side reactions, where glutaraldehyde reacts with other amino acid side chains, cysteine, histidine, and tyrosine. It has also been reported that glutaraldehyde can polymerize in aqueous solution. Newer crosslinkers have been found that are more specific for the amino group, such as the N-hydroxysuccinimide esters, and are commonly preferred for forming conjugates. However, most of these linkers hydrolyze in solution, so that the activity is lost over several hours, whereas the aldehyde group is stable in solution, and may have an advantage of overall efficiency.

Chemically Contaminated Eel Fed to Pregnant and Lactating Mouse Dams Causes Hyperactivity in Their Offspring

2016 ◽  
Vol 86 (1-2) ◽  
pp. 36-47 ◽  
Author(s):  
Imen Dridi ◽  
Nidhal Soualeh ◽  
Torsten Bohn ◽  
Rachid Soulimani ◽  
Jaouad Bouayed
Keyword(s):  
Open Field ◽  
Early Life ◽  
Home Cage ◽  
Anguilla Anguilla ◽  
Acetylcholinesterase Activity ◽  
Female Offspring ◽  
Significant Inhibition ◽  
Standard Diet ◽  
Early Life Exposure ◽  
Cage Test

Abstract.This study examined whether perinatal exposure to polluted eels (Anguilla anguilla L.) induces changes in the locomotor activity of offspring mice across lifespan (post-natal days (PNDs) 47 – 329), using the open field and the home cage activity tests. Dams were exposed during gestation and lactation, through diets enriched in eels naturally contaminated with pollutants including PCBs. Analysis of the eel muscle focused on the six non-dioxin-like (NDL) indicator PCBs (Σ6 NDL-PCBs: 28, 52, 101, 138, 153 and 180). Four groups of dams (n = 10 per group) received either a standard diet without eels or eels (0.8 mg/kg/day) containing 85, 216, or 400 ng/kg/day of ϵ6 NDL-PCBs. The open field test showed that early-life exposure to polluted eels increased locomotion in female offspring of exposed dams but not in males, compared to controls. This hyperlocomotion appeared later in life, at PNDs 195 and 329 (up to 32 % increase, p < 0.05). In addition, overactivity was observed in the home cage test at PND 305: exposed offspring females showed a faster overall locomotion speed (3.6 – 4.2 cm/s) than controls (2.9 cm/s, p <0.05); again, males remained unaffected. Covered distances in the home cage test were only elevated significantly in offspring females exposed to highest PCB concentrations (3411 ± 590 cm vs. 1377 ± 114 cm, p < 0.001). These results suggest that early-life exposure to polluted eels containing dietary contaminants including PCBs caused late, persistent and gender-dependent neurobehavioral hyperactive effects in offspring mice. Furthermore, female hyperactivity was associated with a significant inhibition of acetylcholinesterase activity in the hippocampus and the prefrontal cortex.

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