Ligand-Induced Conformational Changes near the Active Site Regulating Enzyme Activity of Momorcharins from Seeds of Bitter Gourd

Abstract Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

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P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

The Journal of Biochemistry ◽

10.1093/jb/mvaa083 ◽

2020 ◽

Vol 168 (5) ◽

pp. 557-567

Author(s):

Wanitcha Rachadech ◽

Yusuke Kato ◽

Rabab M Abou El-Magd ◽

Yuji Shishido ◽

Soo Hyeon Kim ◽

...

Keyword(s):

Amino Acid ◽

Conformational Changes ◽

Active Site ◽

Structural Changes ◽

Catalytic Efficiency ◽

Acid Oxidase ◽

Wild Type ◽

Amino Acid Oxidase ◽

Adenine Ring ◽

The Impact

Abstract Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.

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Phenoxyethyl Piperidine/Morpholine Derivatives as PAS and CAS Inhibitors of Cholinesterases: Insights for Future Drug Design

Scientific Reports ◽

10.1038/s41598-019-56463-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Yaghoub Pourshojaei ◽

Ardavan Abiri ◽

Khalil Eskandari ◽

Zahra Haghighijoo ◽

Najmeh Edraki ◽

...

Keyword(s):

Conformational Changes ◽

Active Site ◽

Inhibitory Activity ◽

Reference Drug ◽

Catalytic Active Site ◽

Electric Eel ◽

Interesting Candidate ◽

Future Drug ◽

Morpholine Derivatives

AbstractAcetylcholinesterase (AChE) catalyzes the conversion of Aβ peptide to its aggregated form and the peripheral anionic site (PAS) of AChE is mainly involved in this phenomenon. Also catalytic active site (CAS) of donepezil stimulates the break-down of acetylcholine (ACh) and depletion of ACh in cholinergic synapses are well established in brains of patients with AD. In this study, a set of compounds bearing phenoxyethyl amines were synthesized and their inhibitory activity toward electric eel AChE (eeAChE) and equine butyrylcholinesterase (eqBuChE) were evaluated. Molecular dynamics (MD) was employed to record the binding interactions of best compounds against human cholinesterases (hAChE and hBuChE) as well as donepezil as reference drug. In vitro results revealed that compound 5c is capable of inhibiting eeAChE activity at IC50 of 0.50 µM while no inhibitory activity was found for eqBuChE for up to 100 µM concentrations. Compound 5c, also due to its facile synthesis, small structure and high selectivity for eeAChE would be very interesting candidate in forthcoming studies. The main interacting parts of compound 5c and compound 7c (most potent eeAChE and eqBuChE inhibitors respectively) with receptors which confer selectivity for AChE and BuChE inhibition were identified, discussed, and compared with donepezil’s interactions. Also during MD simulation it was discovered for the first time that binding of substrates like donepezil to dual CAS and PAS or solely CAS region might have a suppressive impact on 4-α-helical bundles near the tryptophan amphiphilic tetramerization (WAT) domain of AChE and residues which are far away from AChE active site. The results proposed that residues involved in donepezil interactions (Trp86 and Phe295) which are located in CAS and mid-gorge are the mediator of conformational changes in whole protein structure.

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The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

Biochemical Journal ◽

10.1042/bj3330811 ◽

1998 ◽

Vol 333 (3) ◽

pp. 811-816 ◽

Cited By ~ 14

Author(s):

Antonio PÁRRAGA ◽

Isabel GARCÍA-SÁEZ ◽

Sinead B. WALSH ◽

Timothy J. MANTLE ◽

Miquel COLL

Keyword(s):

Conformational Changes ◽

Active Site ◽

Three Dimensional ◽

Dimensional Structure ◽

Water Molecules ◽

X Ray Diffraction ◽

Glutathione S Transferase ◽

X Ray ◽

The Right

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

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Comparison of the activities of protein disulphide-isomerase and thioredoxin in catalysing disulphide isomerization in a protein substrate

Biochemical Journal ◽

10.1042/bj2750349 ◽

1991 ◽

Vol 275 (2) ◽

pp. 349-353 ◽

Cited By ~ 74

Author(s):

H C Hawkins ◽

E C Blackburn ◽

R B Freedman

Keyword(s):

Enzyme Activity ◽

Active Site ◽

Specific Activity ◽

Disulphide Bond ◽

Protein Disulphide Isomerase ◽

Protein Substrate ◽

Catalytic Domains ◽

Standard Assay

1. The activities of protein disulphide-isomerase (PDI) and thioredoxin in catalysing disulphide bond isomerization in a protein substrate were compared by using the standard assay, namely the re-activation of ‘scrambled’ RNAase. 2. The specific activity of PDI was 25-fold greater than that of thioredoxin. 3. The greater efficiency of PDI compared with thioredoxin is considered to be due more to the presence of multiple catalytic domains in PDI than to differences in their active-site sequences. 4. Data and procedures were defined for expressing enzyme activity in standard units, i.e. mumol of active RNAase generated/min.

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Activity and conformational changes of horseradish peroxidase in trifluoroethanol

Biochemistry and Cell Biology ◽

10.1139/o02-003 ◽

2002 ◽

Vol 80 (2) ◽

pp. 205-213 ◽

Cited By ~ 6

Author(s):

Hong-Wei Zhou ◽

Yan Xu ◽

Hai-Meng Zhou

Keyword(s):

Enzyme Activity ◽

Horseradish Peroxidase ◽

Conformational Changes ◽

Tertiary Structure ◽

Polyacrylamide Gel Electrophoresis ◽

Intrinsic Fluorescence ◽

Two Phase ◽

Main Effect ◽

Α Helix ◽

Kinetics Of

The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 525% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (2540%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The α-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.Key words: horseradish peroxidase, trifluoroethanol, unfolding, Soret.

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