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Site-Specific Chemical Modification of Proteins
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Increased insight into these data has been derived from recent studies on the site-specific mutagenesis of rat trypsin.27,28 In the first of these studies,27 a cysteinyl residue was introduced in place of the active-site serine residue by site-specific mutagenesis and the catalytic activity characterized with various substrates. While ‘thiol-trypsin (trypsin S195C) was active, the value for kcat for trypsin in S195C was reduced by a factor of 3.4 × 105 with respect to the native enzyme. In a subsequent crystallographic study of trypsin S195C, it was determined that there are likely steric considerations, such as blocking of the oxyanion hole by the sulfur atom, that contribute to the low levels of activity.
Multi-Functional Monoamine Oxidase and Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Ireen Denya, Sarel F. Malan, Jacques Joubert
AChE and BuChE share a 65% amino acid sequence homology and have similar molecular forms and active site structure (Allderdice et al., 1991). Early kinetic studies indicate that the active site of AChE contains two subsites, the esteratic and anionic subsites, corresponding to the catalytic and choline–binding pockets, respectively. The anionic active subsite interacts with the charged quaternary group of the choline moiety of acetylcholine (Augustinsson et al., 1950; Rosenberry, 2006). It contains the amino acids Trp 86, Tyr 133, Tyr 337 and Phe 338 and these bind the quaternary trimethylammonium choline moiety of the substrate mainly through π-cation interactions (Harel et al., 1993) positioning the ester optimally at the acylation site. The acyl pocket, responsible for substrate selectivity by preventing access of the larger choline esters is composed of Phe 295 and Phe 297. The oxyanion hole, Gly 121, Gly 122 and Ala 204, provides hydrogen bond donors that stabilise the tetrahedral transition state of the substrate (Ordentlich et al., 1998). Cationic substrates are not bound by a negatively charged amino acid in the anionic site, but by interaction of 14 aromatic residues that line the gorge leading to the active site (Colovic et al., 2013). Among the aromatic amino acids, Trp 84 is reported to be critical and not substitutable (Tougu, 2001). The esteratic subsite or cationic active site (CAS) where ACh is hydrolysed contains a catalytic triad of three amino acids, namely Ser 203, His 447 and Glu 334 (Colovic et al., 2013).
Protein-Based Bioscavengers of Organophosphorus Nerve Agents
Published in Brian J. Lukey, James A. Romano, Salem Harry, Chemical Warfare Agents, 2019
Moshe Goldsmith, Yacov Ashani, Tamara. C. Otto, C. Linn Cadieux, David. S. Riddle
Unlike HuAChE, AChE obtained from the venom of snakes such as Bungarus fasciatus (BfAChE; Uniprot Q92035) (Kumar and Elliott, 1973) can be expressed as a monomeric protein at high levels in mammalian cells (Cousin et al., 1996). This made it an attractive target for protein engineering for the purpose of improving its reactivation rates following OPNA inhibition. For this purpose, researchers introduced a nucleophilic histidine residue into the oxyanion hole of AChE similar to the G117H mutation in HuBChE, which conferred OP hydrolase activity on BChE (Poyot et al., 2006). Since the G122H-BfAChE mutant had completely lost its acetylcholine hydrolase activity, additional mutations were introduced into the enzyme’s active site based on a molecular modeling analysis (Poyot et al., 2006). This analysis suggested that the G122H mutation had distorted the active site by causing steric hindrance. Thus, two additional mutations, Y124Q and S125T, were introduced to increase the space in the active site and to mimic additional BChE residues at this region (Poyot et al., 2006). The resulting triple mutant H122H/Y124Q/S125T-BfAChE displayed a four orders of magnitude reduction in catalytic efficiency with acetylthiocholine with respect to wild type BfAChE but was able to slowly hydrolyze the OP pesticides DFP and paraoxon in addition to OP echothiophate (Poyot et al., 2006). Still, the efficiency of hydrolysis of these OPs was one or two orders of magnitude lower than that of G117H-HuBChE, and the mutant was suggested to resist OP inhibition mostly due to poor OP binding affinities. An attempt to graft the same set of mutations onto HuAChE did not generate an improved variant and was not further studied (Poyot et al., 2006).
An updated patent review of monoacylglycerol lipase (MAGL) inhibitors (2018-present)
Published in Expert Opinion on Therapeutic Patents, 2021
Giulia Bononi, Giulio Poli, Flavio Rizzolio, Tiziano Tuccinardi, Marco Macchia, Filippo Minutolo, Carlotta Granchi
The obtainment of MAGL crystal structures [6,7] has provided an in-depth understanding of the active site and catalytic mechanism of the enzyme. MAGL possesses a long hydrophobic channel, normally hosting the aliphatic chain of the substrate 2-AG, and this tunnel, which can be accessible from the outside by the opening of a flexible lid, ends with a small hydrophilic pocket inside the protein, which is the cavity where the glycerol portion of 2-AG nicely fits. The catalysis is allowed by the catalytic triad, Ser122-Asp239-His269: Ser122 is activated thanks to the basicity of His269, which is involved in a strong hydrogen-bond with Asp239. Ser122 triggers a nucleophilic attack to the carbonyl of the substrate 2-AG, forming a complex with it. The tetrahedral intermediate formed during the transition state is placed in a cavity of the enzyme named ‘oxyanion hole.’ Finally, the enzyme is regenerated due to the hydrolysis of the enzyme-substrate complex mediated by His269, thus releasing arachidonic acid and glycerol. Three cysteine residues (Cys201, Cys208 and Cys242) located near the catalytic triad have a role in the stabilization of the active conformation of MAGL [8].
Design, synthesis and characterization of enzyme-analogue-built polymer catalysts as artificial hydrolases
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Divya Mathew, Benny Thomas, Karakkattu Subrahmanian Devaky
The catalytic process begins with shape selective recognition and binding of the substrate in the active site. The proper binding of the side-chain of the amino acid residue to the recognition site on the enzyme is essential. Then nucleophilic attack by the hydroxyl group of serine leads to the formation of an acyl intermediate. The nucleophilic action of serine is initiated by the proton abstraction by Histidine-57, which is enhanced by the aspartic acid moiety. A covalent bond is formed between the Ser-195 side-chain oxygen and the substrate. The negative charge developed on the peptide carbonyl oxygen is stabilized by hydrogen bonding with amide protons of protease backbone. This region of the protein is called the “oxyanion hole”, because it stabilizes the negative charge on the oxygen; the oxyanion hole is critical for catalysis. His-57 donates a proton to the amide nitrogen of the substrate, leading to the release of the C-terminal part of the substrate as a free peptide. The final step of the catalytic proteolysis is the nucleophilic attack of water molecule on the ester bond between the peptide and the Ser-195 oxygen. A second peptide with one amino acid less gets formed and regenerates the serine hydroxyl for further nucleophilic attack. The second peptide then dissociates from the enzyme to allow another catalytic cycle to begin.
Metformin and its sulphonamide derivative simultaneously potentiateanti-cholinesterase activity of donepezil and inhibit beta-amyloid aggregation
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2018
Magdalena Markowicz-Piasecka, Kristiina M. Huttunen, Joanna Sikora
The crystallographic structure of AChE reveals that it includes two separate ligand binding sites; a PAS at the entrance consisting of Trp86, Tyr337, Trp286, and Tyr72, and a catalytic active site (CAS) at the bottom. An active site of AChE contains 1) an esteratic site (ES) with the catalytic triad Ser200-His440-Glu327; 2) an oxyanion hole (OAH); 3) an acyl binding site (ABS); and 4) an anionic substrate binding site (AS)16,34. Therefore, inhibitors binding to either site CAS or PAS could inhibit AChE47. Donepezil inhibits AChE through binding with the active site by interactions with benzyl substituent (CAS of AChE), the atom of the piperidine (mid-gorge) and dimethoxyindanone moiety (PAS of AChE)47. It has been recently stated that AChE promotes amyloid fibril formation by interaction through the PAS of AchE, therefore, the development of novel agents capable of dual binding (both CAS and PAS) is a very desirable and promising approach.