α-Glucosidase and α-Amylase Inhibitory Compounds from three African Medicinal Plants: An Enzyme Inhibition Kinetics Approach

The quest to find new lead compounds with anti-diabetic effects via the inhibition of α-glucosidase and α-amylase had led us to conduct bioassay guided isolation of three African medicinal plants which resulted in the identification of bicyclo[2.2.0]hexane-2,3,5-triol (1), 3β- O-acetyl betulinic acid (2) and 2,7-dihydroxy-4 H-1-benzopyran-4-one (3), as the bioactive compounds. The compounds demonstrated a significant (P < 0.05) inhibitory effect on α-glucosidase and α-amylase activities than acarbose. Steady state kinetic analysis revealed that compounds 1 and 2 inhibited both α-amylase and α-glucosidase in non-competitive patterns whilst compound 3 was an uncompetitive inhibitor of α-glucosidase and a non-competitive inhibitor of α-amylase. In conclusion, the study has identified three new active α-glucosidase and α-amylase inhibitory compounds that could have the potential to retard postprandial hyperglycemia.

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Interaction of difluoro-oxaloacetate with aspartate transaminase

Biochemical Journal ◽

10.1042/bj1610383 ◽

1977 ◽

Vol 161 (2) ◽

pp. 383-387 ◽

Cited By ~ 8

Author(s):

P A Briley ◽

R Eisenthal ◽

R Harrison ◽

G D Smith

Keyword(s):

Steady State ◽

Competitive Inhibitor ◽

Aspartate Transaminase ◽

Steady State Kinetic ◽

High Concentrations ◽

State Kinetic ◽

Uncompetitive Inhibitor

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.

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α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors

Biochemical Journal ◽

10.1042/bj3600727 ◽

2001 ◽

Vol 360 (3) ◽

pp. 727-736 ◽

Cited By ~ 9

Author(s):

Bernd NIDETZKY ◽

Christian EIS

Keyword(s):

Steady State ◽

Transition State ◽

Schizophyllum Commune ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Competitive Inhibitor ◽

Chemical Mechanism ◽

Steady State Kinetic ◽

State Kinetic ◽

Trehalose Phosphorylase

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme–substrate complexes formed from binary enzyme–phosphate and enzyme–α-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope = 1.14; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite −1 in the enzyme–phosphate complex with a dissociation constant of 56μM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.

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Synthesis of 2-(4-hydroxyphenyl)ethyl 3,4,5-Trihydroxybenzoate and Its Inhibitory Effect on Sucrase and Maltase

Processes ◽

10.3390/pr8121603 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1603

Author(s):

Wen-Tai Li ◽

Yu-Hsuan Chuang ◽

Jiahn-Haur Liao ◽

Jung-Feng Hsieh

Keyword(s):

Active Site ◽

Docking Study ◽

Competitive Inhibitor ◽

Inhibitory Effect ◽

Kinetics Study ◽

Amino Acid Residues ◽

Inhibition Kinetics ◽

Molecular Docking Study ◽

Hydrogen Bond Interactions ◽

Glucosidase Inhibitors

We report on the synthesis of an active component, 2-(4-hydroxyphenyl)ethyl 3,4,5-trihydroxybenzoate (HETB), from Rhodiola crenulata. Subsequent analysis revealed that HETB exhibits α-glucosidase inhibitory activities on maltase and sucrase, with potency exceeding that of the known α-glucosidase inhibitors (voglibose and acarbose). An inhibition kinetics study revealed that HETB, acarbose, and voglibose bind to maltase and sucrase, and HETB was shown to be a strong competitive inhibitor of maltase and sucrase. In a molecular docking study based on the crystal structure of α-glucosidase from Saccharomyces cerevisiae, we revealed the HETB binding in the active site of maltase via hydrogen-bond interactions with five amino acid residues: Ser 240, Asp 242, Glu 277, Arg 315, and Asn 350. For HETB docked to the sucrase active site, seven hydrogen bonds (with Asn 114, Glu 148, Gln 201, Asn 228, Gln 381, Ile 383, and Ser 412) were shown.

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Nitrogenase of Klebsiella pneumoniae. Hydrazine is a product of azide reduction

Biochemical Journal ◽

10.1042/bj1930971 ◽

1981 ◽

Vol 193 (3) ◽

pp. 971-983 ◽

Cited By ~ 29

Author(s):

M J Dilworth ◽

R N F Thorneley

Keyword(s):

Klebsiella Pneumoniae ◽

Electron Flux ◽

Competitive Inhibitor ◽

Spectrometric Analysis ◽

H2 Evolution ◽

Total Electron ◽

Steady State Kinetic ◽

Nitrogen Bonds ◽

State Kinetic ◽

Nitrogen Bond

Klebsiella pneumoniae nitrogenase reduced azide, at 30 degrees C and pH 6.8-8.2, to yield ammonia (NH3), dinitrogen (N2) and hydrazine (N2H4). Reduction of (15N = 14N = 14N)-followed by mass-spectrometric analysis showed that no new nitrogen-nitrogen bonds were formed. During azide reduction, added 15N2H4 did not contribute 15N to NH3, indicating lack of equilibration between enzyme-bound intermediates giving rise to N2H4 and N2H4 in solution. When azide reduction to N2H4 was partially inhibited by 15N2, label appeared in NH3 but not in N2H4. Product balances combined with the labelling data indicate that azide is reduced according to the following equations: (formula: see text); N2 was a competitive inhibitor and CO a non-competitive inhibitor of azide reduction to N2H4. The percentage of total electron flux used for H2 evolution concomitant with azide reduction fell from 26% at pH 6.8 to 0% at pH 8.2. Pre-steady-state kinetic data suggest that N2H4 is formed by the cleavage of the alpha-beta nitrogen-nitrogen bond to bound azide to leave a nitride (= N) intermediate that subsequently yields NH3.

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Steady-state kinetic mechanism of bovine brain tubulin: tyrosine ligase

Biochemical Journal ◽

10.1042/bj2860243 ◽

1992 ◽

Vol 286 (1) ◽

pp. 243-251 ◽

Cited By ~ 9

Author(s):

N L Deans ◽

R D Allison ◽

D L Purich

Keyword(s):

Steady State ◽

Thermal Inactivation ◽

Enzyme Reaction ◽

Kinetic Mechanism ◽

Competitive Inhibitor ◽

Product Inhibition ◽

Maximal Velocity ◽

Steady State Kinetic ◽

Tubulin Tyrosine Ligase ◽

State Kinetic

The ATP-dependent resynthesis of tubulin from tyrosine and untyrosinated tubulin was examined to establish the most probable steady-state kinetic mechanism of the tubulin: tyrosine ligase (ADP-forming). Three pair-wise sets of initial rate experiments, involving variation of two substrates pair-wise with the third substrate held at a high (but non-saturating) level, yielded convergent-line data, a behaviour that is diagnostic for sequential mechanisms. Michaelis constants were 14 microM, 1.9 microM and 17 microM for ATP, untyrosinated tubulin and L-tyrosine respectively, and the maximal velocity was 0.2 microM/min. AMP was a competitive inhibitor with respect to ATP, and a non-competitive inhibitor versus either tubulin or tyrosine. Likewise, L-dihydroxyphenylalanine acted competitively relative to tyrosine and non-competitively with respect to either ATP or tubulin. These findings directly support a random sequential mechanism. Product inhibition patterns with ADP were also consistent with this assignment; however, inhibition studies were not practical with either orthophosphate or tyrosinated tubulin because both were very weak inhibitors. Substrate protection of the enzyme against alkylation by N-ethylmaleimide and thermal inactivation, along with evidence of enzyme binding to ATP-Sepharose and tubulin-Sepharose, also supports the idea that this three-substrate enzyme reaction exhibits a random substrate addition pathway.

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Design, Synthesis, and Evaluation of Acetylcholinesterase and Butyrylcholinesterase Dual-Target Inhibitors against Alzheimer’s Diseases

Molecules ◽

10.3390/molecules25030489 ◽

2020 ◽

Vol 25 (3) ◽

pp. 489

Author(s):

Yan Guo ◽

Hongyu Yang ◽

Zhongwei Huang ◽

Sen Tian ◽

Qihang Li ◽

...

Keyword(s):

Inhibitory Effect ◽

Inhibition Kinetics ◽

Anionic Site ◽

Design Synthesis ◽

Potential Safety ◽

Catalytic Sites ◽

Enzyme Inhibition Kinetics ◽

Molecular Modeling Studies ◽

Inhibitory Activities

A series of novel compounds 6a–h, 8i–1, 10s–v, and 16a–d were synthesized and evaluated, together with the known analogs 11a–f, for their inhibitory activities towards acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The inhibitory activities of AChE and BChE were evaluated in vitro by Ellman method. The results show that some compounds have good inhibitory activity against AChE and BChE. Among them, compound 8i showed the strongest inhibitory effect on both AChE (eeAChE IC50 = 0.39 μM) and BChE (eqBChE IC50 = 0.28 μM). Enzyme inhibition kinetics and molecular modeling studies have shown that compound 8i bind simultaneously to the peripheral anionic site (PAS) and the catalytic sites (CAS) of AChE and BChE. In addition, the cytotoxicity of compound 8i is lower than that of Tacrine, indicating its potential safety as anti-Alzheimer’s disease (anti-AD) agents. In summary, these data suggest that compound 8i is a promising multipotent agent for the treatment of AD.

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Investigation of Some Medicinal Plants Inhibitory Effect on NO Production in Oxidative Stressed PC12 Cells

Pharmaceutical Sciences ◽

10.15171/ps.2019.20 ◽

2019 ◽

Vol 25 (2) ◽

pp. 132-137

Author(s):

Hamed Parsa Khankandi ◽

Sahar Behzad ◽

Shamim Sahranavard ◽

Mina Rezvani ◽

Naghmeh Tadris Hasani

Keyword(s):

Oxidative Stress ◽

Medicinal Plants ◽

Pc12 Cells ◽

Inflammatory Diseases ◽

Inhibitory Effect ◽

No Production ◽

Lead Compounds ◽

Stressed Cells

Background: Nitric oxide and reactive nitrogen species play an important role in various pathological conditions like cancer, inflammation and neurodegeneration. As plants and natural compounds have a great potency of discovering lead compounds which might affect NO production during inflammation and various pathologies, we examined the effects of three medicinal plants native to Iran, on NO production during oxidative stress in PC12 cells. Methods: In this study, cell death and NO levels were measured by MTT and by Griess assay, respectively. Oxidative stress was induced by hydrogen peroxide and extracts of Astragalus jolderensis, Convolvulus commutatus and Salvia multicaulis were used as pretreatment in oxidative stressed PC12 cells. Results: A. jolderensis extract significantly suppressed NO production in 150 and 200 μg/ml concentrations and C. commutatus extract in all concentration inhibited NO production in stressed PC12 cells. In addition, the extract of S. multicaulis inhibited NO production during stress at all concentrations above 50 μg/ml. Besides, the extract of S. multicaulis showed protective effect at lower doses in stressed cells. Conclusion: According to the results, S. multicaulis inhibited NO production and protected cells from oxidative stress. Hence, S. multicaulis is a good candidate for further in vitro and in vivo investigations. A. jolderensis and C. commutatus also suppressed NO production during stress. Therefore, they could be noticed in experiments that centralize on the inhibition of NO production and drug discovery studies in the field of neurodegenerative and chronic inflammatory diseases.

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