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Peripheral Anionic Site (peripheral + anionic_site)
Selected AbstractsHydrolysis of acetylthiocoline, o -nitroacetanilide and o- nitrotrifluoroacetanilide by fetal bovine serum acetylcholinesteraseFEBS JOURNAL, Issue 7 2009María F. Montenegro Besides esterase activity, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze o -nitroacetanilides through aryl acylamidase activity. We have reported that BuChE tetramers and monomers of human blood plasma differ in o -nitroacetanilide (ONA) hydrolysis. The homology in quaternary structure and folding of subunits in the prevalent BuChE species () of human plasma and AChE forms of fetal bovine serum prompted us to study the esterase and amidase activities of fetal bovine serum AChE. The kcat/Km values for acetylthiocholine (ATCh), ONA and its trifluoro derivative N -(2-nitrophenyl)-trifluoroacetamide (F-ONA) were 398 × 106 m,1·min,1, 0.8 × 106 m,1·min,1, and 17.5 × 106 m,1·min,1, respectively. The lack of inhibition of amidase activity at high F-ONA concentrations makes it unlikely that there is a role for the peripheral anionic site (PAS) in F-ONA degradation, but the inhibition of ATCh, ONA and F-ONA hydrolysis by the PAS ligand fasciculin-2 points to the transit of o -nitroacetalinides near the PAS on their way to the active site. Sedimentation analysis confirmed substrate hydrolysis by tetrameric 10.9S AChE. As compared with esterase activity, amidase activity was less sensitive to guanidine hydrochloride. This reagent led to the formation of 9.3S tetramers with partially unfolded subunits. Their capacity to hydrolyze ATCh and F-ONA revealed that, despite the conformational change, the active site architecture and functionality of AChE were partially retained. [source] Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous estersFEBS JOURNAL, Issue 10 2008Patrick Masson The effects of tyramine, serotonin and benzalkonium on the esterase and aryl acylamidase activities of wild-type human butyrylcholinesterase and its peripheral anionic site mutant, D70G, were investigated. The kinetic study was carried out under steady-state conditions with neutral and positively charged aryl acylamides [o -nitrophenylacetanilide, o -nitrotrifluorophenylacetanilide and m -(acetamido) N,N,N -trimethylanilinium] and homologous esters (o -nitrophenyl acetate and acetylthiocholine). Tyramine was an activator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for positively charged substrates. The affinity of D70G for tyramine was lower than that of the wild-type enzyme. Tyramine activation of hydrolysis for neutral substrates by D70G was linear. Tyramine was found to be a pure competitive inhibitor of hydrolysis for positively charged substrates with both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both esterase and aryl acylamidase activities for both positively charged and neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyperbolic (i.e. partial) with neutral substrates and linear with positively charged substrates. Inhibition of D70G was linear with all substrates. A comparison of the effects of tyramine and serotonin on D70G versus the wild-type enzyme indicated that: (a) the peripheral anionic site is involved in the nonlinear activation and inhibition of the wild-type enzyme; and (b) in the presence of charged substrates, the ligand does not bind to the peripheral anionic site, so that ligand effects are linear, reflecting their sole interaction with the active site binding locus. Benzalkonium acted as an activator at low concentrations with neutral substrates. High concentrations of benzalkonium caused parabolic inhibition of the activity with neutral substrates for both wild-type butyrylcholinesterase and D70G, suggesting multiple binding sites. Benzalkonium caused linear, noncompetitive inhibition of the positively charged aryl acetanilide m -(acetamido) N,N,N -trimethylanilinium for D70G, and an unusual mixed-type inhibition/activation (, > , > 1) for wild-type butyrylcholinesterase with this substrate. No fundamental difference was observed between the effects of ligands on the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus, butyrylcholinesterase uses the same machinery, i.e. the catalytic triad S198/H448/E325, for the hydrolysis of both types of substrate. The differences in response to ligand binding depend on whether the substrates are neutral or positively charged, i.e. the differences depend on the function of the peripheral site in wild-type butyrylcholinesterase, or the absence of its function in the D70G mutant. The complex inhibition/activation effects of effectors, depending on the integrity of the peripheral anionic site, reflect the allosteric ,cross-talk' between the peripheral anionic site and the catalytic centre. [source] Investigations into the development of catalytic activity in anti-acetylcholinesterase idiotypic and anti-idiotypic antibodiesJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2009Glynis Johnson Abstract We have previously described anti-acetylcholinesterase antibodies that display acetylcholinesterase-like catalytic activity. No evidence of contaminating enzymes was found, and the antibodies are kinetically and apparently structurally distinct from both acetylcholinesterase (AChE) and butyrylcholinesterase. We have also mimicked the antibody catalytic sites in anti-anti-idiotypic (Ab3) antibodies. Independently from us, similar acetylcholinesterase-like antibodies have been raised as anti-idiotypic (Ab2) antibodies against a non-catalytic anti-acetylcholinesterase antibody, AE-2. In this paper, we describe an epitope analysis, using synthetic peptides in ELISA and competition ELISA, and a peptide array, of five catalytic anti-acetylcholinesterase antibodies (Ab1s), three catalytic Ab3s, as well as antibody AE-2 and a non-catalytic Ab2. The catalytic Ab1s and Ab3s recognized three Pro- and Gly-containing sequences (40PPMGPRRFL, 78PGFEGTE, and 258PPGGTGGNDTELVAC) on the AChE surface. As these sequences do not adjoin in the AChE structure, recognition would appear to be due to cross-reaction. This was confirmed by the observation that the sequences superimpose structurally. The non-catalytic antibodies, AE-2 and the Ab2, recognized AChE's peripheral anionic site (PAS), in particular, the sequence 70YQYVD, which contains two of the site's residues. The crystal structure of the AChE tetramer (Bourne et al., 1999) shows direct interaction and high complementarity between the 257CPPGGTGGNDTELVAC sequence and the PAS. Antibodies recognizing the sequence and the PAS may, in turn, be complementary; this may account for the apparent paradox of catalytic development in both Ab1s and Ab2s. The PAS binds, but does not hydrolyze, substrate. The catalytic Ab1s, therefore, recognize a site that may function as a substrate analog, and this, together with the presence of an Arg-Glu salt bridge in the epitope, suggests mechanisms whereby catalytic activity may have developed. In conclusion, the development of AChE-like catalytic activity in anti-AChE Ab1s and Ab2s appears to be the result of a combination of structural complementarity to a substrate-binding site, charge complementarity to a salt bridge, and specific structural peculiarities of the AChE molecule. Copyright © 2008 John Wiley & Sons, Ltd. [source] Selective enhancement of the activity of C-terminally truncated, but not intact, acetylcholinesteraseJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Martina Zimmermann Abstract Acetylcholinesterase (AChE) is one of the fastest enzymes approaching the catalytic limit of enzyme activity. The enzyme is involved in the terminal breakdown of the neurotransmitter acetylcholine, but non-enzymatic roles have also been described for the entire AChE molecule and its isolated C-terminal sequences. These non-cholinergic functions have been attributed to both the developmental and degenerative situation: the major form of AChE present in these conditions is monomeric. Moreover, AChE has been shown to lose its typical characteristic of substrate inhibition in both development and degeneration. This study characterizes a form of AChE truncated after amino acid 548 (T548-AChE), whose truncation site is homologue to that of a physiological form of T-AChE detected in fetal bovine serum that has lost its C-terminal moiety supposedly due to proteolytic cleavage. Peptide sequences covered by this C-terminal sequence have been shown to be crucially involved in both developmental and degenerative mechanisms in vitro. Numerous studies have addressed the structure,function relationship of the AChE C-terminus with T548-AChE representing one of the most frequently studied forms of truncated AChE. In this study, we provide new insight into the understanding of the functional characteristics that T548-AChE acquires in solution: T548-AChE is incubated with agents of varying net charge and molecular weight. Together with kinetic studies and an analysis of different molecular forms and aggregation states of T548-AChE, we show that the enzymatic activity of T548-AChE, an enzyme verging at its catalytic limit is, nonetheless, apparently enhanced by up to 800%. We demonstrate, first, how the activity of T548-AChE can be enhanced through agents that contain highly positive charged moieties. Moreover, the un-competitive mechanism of activity enhancement most likely involves the peripheral anionic site of AChE that is reflected in delayed substrate inhibition being observed for activity enhanced T548-AChE. The data provides evidence towards a mechanistic and functional link between the form of AChE unique to both development and degeneration and a C-terminal peptide of T-AChE acting under those conditions. [source] Tacrine,Melatonin Hybrids as Multifunctional Agents for Alzheimer's Disease, with Cholinergic, Antioxidant, and Neuroprotective PropertiesCHEMMEDCHEM, Issue 5 2009María Isabel Fernández-Bachiller Dr. Abstract Tacrine,melatonin hybrids are potential multifunctional drugs for Alzheimer's disease that may simultaneously palliate intellectual deficits and protect the brain against both ,-amyloid peptide and oxidative stress. Molecular modeling studies show that they target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. They are nontoxic and may be able to penetrate the CNS, according to in,vitro PAMPA-BBB assays. Tacrine,melatonin hybrids were designed and synthesized as new multifunctional drug candidates for Alzheimer's disease. These compounds may simultaneously palliate intellectual deficits and protect the brain against both ,-amyloid (A,) peptide and oxidative stress. They show improved cholinergic and antioxidant properties, and are more potent and selective inhibitors of human acetylcholinesterase (hAChE) than tacrine. They also capture free radicals better than melatonin. Molecular modeling studies show that these hybrids target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. At sub-micromolar concentrations they efficiently displace the binding of propidium iodide from the PAS and could thus inhibit A, peptide aggregation promoted by AChE. Moreover, they also inhibit A, self-aggregation and display neuroprotective properties in a human neuroblastoma line against cell death induced by various toxic insults, such as A,25,35, H2O2, and rotenone. Finally, they exhibit low toxicity and may be able to penetrate the central nervous system according to an in,vitro parallel artificial membrane permeability assay for the blood,brain barrier (PAMPA-BBB). [source] | ||||||||||