RESUMO
Flavonoids are recognized to regulate animals' food digestion processes trough interaction with digestive enzymes. The binding capacity of hesperetin (HES), luteolin (LUT), quercetin (QUE), catechin (CAT) and rutin (RUT) with pancreatic α-amylase were evaluated, using UV-Vis spectroscopy, fluorescence and molecular docking. Using p-nitrophenyl-α-d-maltopentoside (pNPG5) as substrate analog, LUT showed the best inhibitory capacity, even better than that of the positive control, acarbose (ACA). A mixed-type inhibition was observed for HES, LUT and QUE, a competitive-type for ACA, while no inhibition was observed with CAT and RUT. In agreement with kinetic results, α-amylase presented a higher affinity for LUT, when analyzed by fluorescence quenching. The binding of flavonoids to amylase followed a static mechanism, where the binding of one flavonoid per enzyme molecule was observed. Docking analysis showed that flavonoids bound near to enzyme active site, while ACA bound in another site behind the catalytic triad. Extrinsic fluorescence analysis, together with docking analysis pointed out that hydrophobic interactions regulated the flavonoid-α-amylase interactions. The present study provides evidence to understand the relationship of flavonoids structure with their inhibition mechanism.
Assuntos
Flavonoides/química , Flavonoides/farmacologia , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/química , Sítios de Ligação , Flavonoides/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Relação Estrutura-Atividade , alfa-Amilases/metabolismoRESUMO
Glucosamine-6P-deaminase (EC 3.5.99.6, formerly glucosamine-6-phosphate isomerase, EC 5.3.1.10) from Escherichia coli is an attractive experimental model for the study of allosteric transitions because it is both kinetically and structurally well-known, and follows rapid equilibrium random kinetics, so that the kinetic K(m) values are true thermodynamic equilibrium constants. The enzyme is a typical allosteric K-system activated by N-acetylglucosamine 6-P and displays an allosteric behavior that can be well described by the Monod-Wyman-Changeux model. This thermodynamic study based on the temperature dependence of allosteric parameters derived from this model shows that substrate binding and allosteric transition are both entropy-driven processes in E. coli GlcN6P deaminase. The analysis of this result in the light of the crystallographic structure of the enzyme implicates the active-site lid as the structural motif that could contribute significantly to this entropic component of the allosteric transition because of the remarkable change in its crystallographic B factors.