a-Amylase Inhibition, Anti-glycation Property and Characterization of the Binding Interaction of Citric Acid with a-Amylase Using Multiple Spectroscopic, Kinetics and Molecular Docking Approaches
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Date
2022-07-15
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Elsevier
Abstract
The quest to suppress complications associated with diabetes mellitus is ever increasing, while food additives
and preservatives are currently being considered to play additional roles besides their uses in food
enhancement and preservation. In the present study, the protective prowess of a common food preservative
(citric acid, CA) against advanced glycation end-products (AGEs) formation and its binding interaction
mechanism with a-amylase (AMY), an enzyme linked with hyperglycemia management, were
examined. Enzyme inhibition kinetics, intrinsic fluorescence, synchronous and 3D fluorescence spectroscopies,
ultraviolet–visible (UV–Vis) absorption spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy,
thermodynamics, and molecular docking analyses were employed. Results obtained showed
that citric acid decreased a-amylase activity via mixed inhibition (IC50 = 5.01 ± 0.87 mM,
Kic = 2.42 mM, Kiu = 160.34 mM) and suppressed AGEs formation (IC50 = 0.795 ± 0.001 mM). The intrinsic
fluorescence of free a-amylase was quenched via static mechanism with high bimolecular quenching
constant (Kq) and binding constant (Ka) values. Analysis of thermodynamic properties revealed that
AMY-CA complex was spontaneously formed (DG < 0), entropy driven (TDS > DH), with involvement
of electrostatic forces. UV–Vis, FT-IR and 3D fluorescence spectroscopies affirmed alterations in aamylase
native conformation due to CA binding interaction. CA interacted with His-101, Asp-197, His-
299, and Glu-233 within AMY active site. Our findings indicated that CA could impair formation of
AGEs and interact with a-amylase to slow down starch hydrolysis; vital properties in management of
type 2 diabetes complications.
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Keywords
a-Amylase inhibition, Citric acid, Anti-glycation, Hyperglycemia, Spectroscopy, Molecular docking