Structural insights into substrate traffic and inhibition in acetylcholinesterase

SynopsisA successful prescription is presented for acetylcholinesterase physically adsorbed on to a mesoporous silicon surface, with a promising hydrolytic response towards acetylthiocholine iodide. The catalytic behaviour of the immobilized enzyme was assessed by spectrophotometric bioassay using neostigmine methyl sulfate as a standard acetycholinesterase inhibitor. The surface modification was studied through field emission SEM, Fourier transform IR spectroscopy, energy-dispersive X-ray spectroscopy, cathode luminescence and X-ray photoelectron spectroscopy analysis, photoluminescence measurement and spectrophotometric bioassay. The porous silicon-immobilized enzyme not only yielded greater enzyme stability, but also significantly improved the native photoluminescence at room temperature of the bare porous silicon architecture. The results indicated the promising catalytic behaviour of immobilized enzyme compared with that of its free counterpart, with a greater stability, and that it aided reusability and easy separation from the reaction mixture. The porous silicon-immobilized enzyme was found to retain 50 % of its activity, promising thermal stability up to 90 • C, reusability for up to three cycles, pH stability over a broad pH of 4-9 and a shelf-life of 44 days, with an optimal hydrolytic response towards acetylthiocholine iodide at variable drug concentrations. On the basis of these findings, it was believed that the porous silicon-immobilized enzyme could be exploited as a reusable biocatalyst and for screening of acetylcholinesterase inhibitors from crude plant extracts and synthesized organic compounds. Moreover, the immobilized enzyme could offer a great deal as a viable biocatalyst in bioprocessing for the chemical and pharmaceutical industries, and bioremediation to enhance productivity and robustness.

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“…spectrophotometric bioassay utilizing neostigmine methyl sulfate as a standard acetylcholinesterase inhibitor [59][60][61].…”

Section: Resultsmentioning

confidence: 99%

Acetylcholinesterase immobilization and characterization, and comparison of the activity of the porous silicon-immobilized enzyme with its free counterpart

et al. 2016

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“…A solution for this problem would be to have ACHe be inhibited by its substrate ACH so that when ACH is at high concentrations the degradation proceeds relatively slowly and then accelerates as the concentration drops. Substrate inhibition of ACHe by ACH was noticed as long ago as 1969 [31], but its functional importance has been emphasized only recently [28,32]. For this scenario to work, ACH should be released very rapidly into the cleft so that the concentration of ACH rises quickly into the inhibitory range.…”

Section: C Reed Et Almentioning

confidence: 99%

“…The peak of the ACHe velocity curve occurs at about 1 mM [28]. The rise time of ACH release is less than 100 ms [26,33], and the concentration of ACH is quickly driven past 1 mM and rises to 10 mM and perhaps higher [32]. This is possible because of the very high concentration of ACH in presynaptic vesicles, approximately 1 M [34].…”

Section: C Reed Et Almentioning

confidence: 99%

The biological significance of substrate inhibition: A mechanism with diverse functions

2010

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Many enzymes are inhibited by their own substrates, leading to velocity curves that rise to a maximum and then descend as the substrate concentration increases. Substrate inhibition is often regarded as a biochemical oddity and experimental annoyance. We show, using several case studies, that substrate inhibition often has important biological functions. In each case we discuss, the biological significance is different. Substrate inhibition of tyrosine hydroxylase results in a steady synthesis of dopamine despite large fluctuations in tyrosine due to meals. Substrate inhibition of acetylcholinesterase enhances the neural signal and allows rapid signal termination. Substrate inhibition of phosphofructokinase ensures that resources are not devoted to manufacturing ATP when it is plentiful. In folate metabolism, substrate inhibition maintains reactions rates in the face of substantial folate deprivation. Substrate inhibition of DNA methyltransferase serves to faithfully copy DNA methylation patterns when cells divide while preventing de novo methylation of methyl-free promoter regions. Keywords:.b iological function; enzyme kinetics; substrate inhibitionThe kinetics of an enzymatic reaction are typically studied by varying the concentration of substrate and plotting the rate of product formation as a function of substrate concentration. In the conventional case this yields a typical hyperbolic Michaelis-Menten curve, and a linear reciprocal LineweaverBurk plot, from which the kinetic constants of the enzyme can be calculated. A surprisingly large number of enzymes do not behave in this conventional way. Instead, their velocity curves rise to a maximum and then decline as the substrate concentration goes up. This phenomenon is referred to as substrate inhibition, and it is estimated that it occurs in some 20% of enzymes [1]. A partial list of enzymes that show substrate inhibition appears in Box 1.Substrate inhibition is often interpreted as an abnormality that comes from using artificially high substrate concentration in a laboratory setting. In a review article on the mechanisms of substrate inhibition in 1994, Kuehl [2] commented that ''although recognized early on as an almost universal phenomenon, it has nevertheless met an almost universal disinterest. Probably the main reason for this neglect is that the majority of enzymologists and many authorities in the field regard substrate inhibition as being almost always a nonphysiological phenomenon.'' There are several reasons for suspecting that substrate inhibition is not a pathological phenomenon, but a biologically relevant regulatory mechanism. First, in many cases normal substrate concentrations are to the right of the velocity maximum, which indicates that these enzymes typically operate under substrate inhibition. Second, many enzymes have specialized sites where a second substrate molecule can bind and act as an allosteric inhibitor. For those enzymes, substrate inhibition is clearly a specially evolved property. Third, evidence is accumulating that substr...

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“…Most of the administered drugs interact with the CT of AChE [15,16]. Furthermore, their interaction with residues (Trp86 and Tyr337) of catalytic anionic subsite (CAS), placed in close proximity to the active site, influences the hydrolysis of ACh [9,13,[17][18][19]. Besides the CT and CAS, the residues of peripheral anionic site (PAS; Tyr72, Asp74, Tyr124, Trp286, and Tyr341) also influence the function of AChE through their allosteric effects [16,20,21].…”

Section: Introductionmentioning

confidence: 99%

“…Besides the CT and CAS, the residues of peripheral anionic site (PAS; Tyr72, Asp74, Tyr124, Trp286, and Tyr341) also influence the function of AChE through their allosteric effects [16,20,21]. Residues of PAS trap the substrate molecules as they enter the gorge, and are involved in accelerating the transfer of the substrate acyl group to a serine hydroxyl group in CAS [17,18,[22][23][24][25]. In addition, PAS site is potentially responsible for colocalization of amyloid-β (Aβ) peptide in the brains of AD patients and has been shown to accelerate the assembly of Aβ to form amyloid fibrils [26][27][28].…”

Section: Introductionmentioning

confidence: 99%

Synthesis and in silico evaluation of 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives against Alzheimer disease: to understand their interacting mechanism with acetylcholinesterase

et al. 2012

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Anomalous action of human acetylcholinesterase (hAChE) in Alzheimer's disease (AD) was restrained by various AChE inhibitors, of which the specific and potent lead candidate Donepezil is used for treating the disease AD. Besides the specificity, the observed undesirable side effects caused by Donepezil invoked the quest for new lead molecules with the increased potency and specificity for AChE. The present study elucidates the potency of six 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives to form a specific interaction with the peripheral anionic site and catalytic anionic subsite residues of hAChE. The NMSMs were prepared in good yield from 1,1-di(methylsulfanyl)-2-nitroethylene and primary amine (or) amino acid esters. In silico interaction analysis reveals specific and potent interactions between hAChE and selected ligand molecules. The sitespecific interactions formed between these molecules also results in a conformational change in the orientation of active site residues of hAChE, which prevents them from being accessed by beta-amyloid protein (Aβ), which is a causative agent for amyloid plaque formation and acetylcholine (ACh). In silico interaction analysis between the ligand-bounded hAChE with Aß and ACh confirms this observation. The variation in the conformation of hAChE associated with the decreased ability of Aβ and ACh to access the respective functional residues of hAChE induced by the novel NMSMs favors their selection for in vivo analysis to present themselves as new members of hAChE inhibitors.

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Structural insights into substrate traffic and inhibition in acetylcholinesterase

Cited by 170 publications

References 61 publications

Acetylcholinesterase immobilization and characterization, and comparison of the activity of the porous silicon-immobilized enzyme with its free counterpart

Acetylcholinesterase immobilization and characterization, and comparison of the activity of the porous silicon-immobilized enzyme with its free counterpart

The biological significance of substrate inhibition: A mechanism with diverse functions

Synthesis and in silico evaluation of 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives against Alzheimer disease: to understand their interacting mechanism with acetylcholinesterase

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