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1.
Int J Biol Macromol ; 113: 212-218, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29477543

RESUMO

Inhibition of α-glucosidase is directly associated with treatment of type 2 diabetes. In this regard, we conducted enzyme kinetics integrated with computational docking simulation to assess the inhibitory effect of raspberry ketone (RK) on α-glucosidase. RK bound to the active site of α-glucosidase and interacted with several key residues such as ASP68, TYR71, HIS111, PHE157, PHE158, PHE177, GLN181, ASP214, THR215, ASP349, ASP408, and ARG439, as detected by protein-ligand docking simulation. Subsequently, we confirmed the action of RK on α-glucosidase as the non-competitive type of inhibition in a reversible and rapidly binding manner. The relevant kinetic parameters were IC50=6.17±0.46mM and Ki=7.939±0.211mM. Regarding the structure-activity relationship, the higher concentration of RK induced slight modulation of the shape of the active site as monitored by hydrophobic exposure. The tertiary conformational change was linked to RK inhibition, and mostly involved regional changes of the active site. Our study provides insight into the functional role of RK due to its structural property of a hydroxyphenyl ring that interacts with the active site of α-glucosidase. We suggest that similar hydroxyphenyl ring compounds targeting the key residues of the active site might be potential α-glucosidase inhibitors.


Assuntos
Butanonas/metabolismo , Butanonas/farmacologia , Inibidores de Glicosídeo Hidrolases/metabolismo , Inibidores de Glicosídeo Hidrolases/farmacologia , Simulação de Acoplamento Molecular , alfa-Glucosidases/metabolismo , Cinética , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , alfa-Glucosidases/química
2.
Int J Biol Macromol ; 112: 686-693, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29425876

RESUMO

In this study we conducted serial kinetic studies integrated with computational simulations to judge the inhibitory effect of pyrogallol on α-glucosidase, due to the association between this enzyme and the treatment of type 2 diabetes. As a result, we found that pyrogallol bound to the active site of α-glucosidase, interacting with several key residues, such as ASP68, MET69, TYR71, PHE157, PHE158, PHE177, GLN181, HIS348, ASP349, ASP406, VAL407, ASP408, ARG439, and ARG443, which was predicted by performing a protein-ligand docking simulation. Subsequently, we evaluated the inhibitory effect of pyrogallol on α-glucosidase, and found that it induced a mixed type of inhibition in a reversible and quick-binding manner. The relevant kinetic parameters were evaluated to be: IC50=0.72±0.051mM; Ki=0.37±0.018mM. A tertiary conformational change was synchronized with pyrogallol inhibition and modulation of the shape of the active site was correspondingly observed. Our study provides insight into the functional inhibitory role of pyrogallol, which results from its triple-hydroxyl groups interacting with the active site of α-glucosidase. We suggest that compounds similar to pyrogallol (phenolic hydroxyl compounds) which target the key residues of the active site of α-glucosidase could be potential agents for α-glucosidase inhibition.


Assuntos
Simulação de Acoplamento Molecular , Pirogalol/farmacologia , Saccharomyces cerevisiae/enzimologia , alfa-Glucosidases/farmacologia , Sítios de Ligação , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Cinética , Ligantes , Naftalenossulfonatos/química , Pirogalol/química , Espectrometria de Fluorescência , Fatores de Tempo , alfa-Glucosidases/química
3.
Int J Biol Macromol ; 103: 829-838, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28539267

RESUMO

Due to the finding that inhibition of α-glucosidase is directly associated with treatment of several diseases, the development of a selective inhibitor for targeting α-glucosidase is important. Gallotannin (GT) is a natural ingredient that has been used as a food additive and for medicinal applications. In this study, we performed a computational docking experiment involving the pre-simulation of the binding mechanism of GT, and the effect of GT on α-glucosidase was evaluated with inhibitory kinetics based on its polyphenol properties. The computational simulations indicated that the hydroxyl groups of GT interact with several residues near the α-glucosidase active site (Met69, Tyr71, Phe177, Arg212, Asp214, Glu276, His348, Asp349, and Arg439), which could affect the catalytic function of α-glucosidase by retarding substrate access. Subsequent kinetic experiments showed that GT conspicuously inhibited α-glucosidase in a parabolic mixed-type manner (IC50=1.31±0.03µM;Ki=0.41±0.032µM). Our study provides insight into the inhibition mechanism and binding manner of GT to α-glucosidase. Based on its α-glucosidase-inhibiting effect and its demonstrated safety as a naturally derived compound, GT represents a promising potential agent for treatment of α-glucosidase-associated diseases.


Assuntos
Inibidores de Glicosídeo Hidrolases/farmacologia , Taninos Hidrolisáveis/farmacologia , alfa-Glucosidases/metabolismo , Inibidores de Glicosídeo Hidrolases/metabolismo , Taninos Hidrolisáveis/metabolismo , Cinética , Simulação de Acoplamento Molecular , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , alfa-Glucosidases/química
4.
Int J Biol Macromol ; 94(Pt B): 836-844, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27629588

RESUMO

Cadmium ions (Cd2+) are a widespread and easily absorbed toxin to both humans and animals that can be spread via food, water, and air pollution. Tyrosinase (EC 1.14.18.1) is a multifunctional copper-containing enzyme that is ubiquitously expressed in animals and plays a critical role in melanin production. We evaluated the toxic effects of Cd2+ on tyrosinase activity and conformation by measuring kinetics and computationally simulating the interactions. We found Cd2+ to be a slope-hyperbolic noncompetitive-inhibition reversible inhibitor of tyrosinase, with an IC50 of 2.92±0.16mM and Ki of 0.23±0.02mM. Spectrofluorimetric measurements of intrinsic and ANS-binding fluorescence showed that Cd2+ did not induce significant changes to tyrosinase overall or to its regional active site conformations. Cd2+ showed its inactivation effect not by modulating apparent structural changes to tyrosinase, but by occupying binding sites. To gain further insight into the Cd2+/tyrosinase interaction, we performed computational docking and molecular dynamics simulations. The results consistently indicated that Cd2+ can interact with several residues near the tyrosinase active site, primarily HIS85 and ASN260. Our study provides insight into the mechanism of the toxic effects Cd2+ has on tyrosinase, which could affect the normal pigmentation pathway in animals.


Assuntos
Cádmio/química , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/química , Asparagina/química , Domínio Catalítico , Cátions Bivalentes , Histidina/química , Humanos , Cinética , Simulação de Dinâmica Molecular , Monofenol Mono-Oxigenase/antagonistas & inibidores , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química
5.
Protein J ; 35(3): 218-24, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27145787

RESUMO

α-Glucosidase is a critical metabolic enzyme that produces glucose molecules by catalyzing carbohydrates. The aim of this study is to elucidate biological toxicity of Cd(2+) based on α-glucosidase activity and conformational changes. We studied Cd(2+)-mediated inactivation as well as conformational modulation of α-glucosidase by using kinetics coupled with simulation of molecular dynamics. The enzyme was significantly inactivated by Cd(2+) in a reversibly binding behavior, and Cd(2+) binding induced a non-competitive type of inhibition reaction (the K i was calculated as 0.3863 ± 0.033 mM). Cd(2+) also modulated regional denaturation of the active site pocket as well as overall partial tertiary structural change. In computational simulations using molecular dynamics, simulated introduction of Cd(2+) induced in a depletion of secondary structure by docking Cd(2+) near the saccharides degradation at the active site, suggesting that Cd(2+) modulating enzyme denaturation. The present study elucidated that the binding of Cd(2+) triggers conformational changes of α-glucosidase as well as inactivates catalytic function, and thus suggests an explanation of the deleterious effects of Cd(2+) on α-glucosidase.


Assuntos
Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Inibidores de Glicosídeo Hidrolases/toxicidade , Saccharomyces cerevisiae/enzimologia , alfa-Glucosidases/metabolismo , Cinética , Simulação de Dinâmica Molecular , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , alfa-Glucosidases/química
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