Binding interaction between (−)-epigallocatechin-3-gallate (EGCG) of green tea and pepsin

Abstract

Analysis of the binding interaction of (−)-epigallocatechin-3-gallate (EGCG) and pepsin is important for understanding the inhibition of digestive enzymes by tea polyphenols. We studied the binding of EGCG to pepsin using fl uorescence spectroscopy, Fourier transform infrared spectroscopy, isothermal titration calorimetry, and protein-ligand docking. We found that EGCG could inhibit pepsin activity. According to thermodynamic parameters, a negative ΔG indicated that the interaction between EGCG and pepsin was spontaneous, and the electrostatic force accompanied by hydrophobic binding forces may play major role in the binding. Data from multi-spectroscopy and docking studies suggest that EGCG could bind pepsin with a change in the native conformation of pepsin. Our results provide further understanding of the nature of the binding interactions between catechins and digestive enzymes.Keywords: EGCG, green tea, pepsin, digestion, interactionGreen tea has many potential health benefi ts attributed to tea polyphenols, in particular catechins, which have strong antioxidative activity (SABU et al., 2010). Major te a catechins include (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin, (−)-epicatechingallate and (−)-epicatechin. EGCG is the most potent constituent of green tea and represents 50% of the total catechins (KILMARTIN & HSU, 2003). Tea polyphenols are soluble in water and can be added to various foods as natural antioxidants. However, tea polyphenols may reduce food digestibility by inhibiting digestive enzymes. Studies have shown that phenolic compounds from plants have a strong ability to interact with food proteins and show strong complexing abilities with enzymes (SPENCER et al., 1988;DUBEAU et al., 2010;YUKSEL et al., 2010;BANDYOPADHYAY et al., 2012). The binding interactions of tea catechins with digestive enzymes were found to denature the catalytic activity of enzymes, which reduced food digestibility (HE et al., 2006). Pepsin is a proteinase present in animals and has a signifi cant role in the digestive process. Therefore, studying the interactions of EGCG and pepsin are important to understand its mechanism of action to inhibit pepsin.Pepsin contains 2 tryptophans (Trp) and 4 tyrosines (Tyr) therefore, fl uorescence quenching can be used with the intrinsic tryptophan of Trp and Tyr to determine the binding affi nities of EGCG and pepsin and characterise the nature of the EGCG-pepsin complex.In this study, we detected the inhibitory effect of EGCG on pepsin activity by studying the interaction of EGCG and pepsin with fl uorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and isothermal titration calorimetry (ITC). To substantiate our experimental studies, docking studies were performed. Important information, such as the change in protein secondary structure and association constant, are reported. We aimed to help understand the nature of binding between catechins and digestive enzymes.

The Effect of Balsamic Vinegar Dressing on Protein and Carbohydrate Digestibility is Dependent on the Food Matrix

“…Comparing different vinegars from grains and fruits, Noh et al observed a decrease in α-amylase activity, the inhibitory effect being higher in vinegars with high organic acid and phenolic content [ 36 ]. Although an inhibitory effect of some polyphenols has been reported also on pepsin activity [ 37 , 38 , 39 , 40 ], and balsamic vinegar possesses the highest concentration of polyphenols among fruit vinegars [ 41 ], we did not observe any modification in trypsin activity. Monomers of tannins such as catechins and gallic acid have been shown to be 1000 times less active in the inhibition of trypsin compared with high-molecular-weight tannic acid [ 42 ].…”

Investigation on the Interaction Behavior Between Oenothein B and Pepsin by Isothermal Titration Calorimetry and Spectral Studies

“…As shown in Table 2, the values of H and S of the interaction between OeB and pepsin at different temperatures are negative, and both increase with the increase of temperature, which are consistent with the characteristic symbols of hydrogen bond, indicating the energy associated with hydrogen bonding dominated whereas the energy associated with hydrophobic interactions was relatively small. Similar behavior is reported in the literature for interaction studies of small molecules polyphenol and pepsin (Li et al, 2016;Y.-Q. Wang & H.-M. Zhang, 2013), which indicates that molecular weight and molecular flexibility have little effect on the type of interaction force between polyphenols and pepsin.…”

“…In other CD studies on small molecule polyphenols and pepsin (Li et al, 2016;Ying et al, 2015), the addition of small molecule polyphenols also reduced the amount of β-sheet, both parallel and antiparallel, in pepsin (EGCG decreased β-sheet from 35.2% to 30.2%, whereas curcumin reduced it from 54.5% to 45.3%), but this decrease was much higher than the OeB-mediated decrease in pepsin β-sheet. Therefore, OeB can have a certain effect on the secondary structure of pepsin, but its effect is less than the effect of small molecule polyphenols on pepsin, which may be due to the weaker hydrophobic interaction between pepsin and OeB.…”

“…Therefore, the addition of OeB may affect the enzyme activity site of pepsin, resulting in a decrease in enzyme activity, and may also bind to the substrate, further preventing the enzymatic hydrolysis of the enzyme–substrate complex by pepsin. The literature reports different behaviors of enzyme kinetics studies of small molecule polyphenols, such as pentagalloyl glucose (PGG) and (‐)‐epigallocatechin gallate (EGCG; Li et al., ; Wu et al., ). The inhibition type of small polyphenols is noncompetitive inhibitors, whereas OeB is a mixture of competitive and noncompetitive inhibitors.…”

“…The point where the line intersects the y ‐axis represents 1/ V max , and the point that intersects the x ‐axis represents 1/ K m , where K m is the Michaelis constant and V max is the maximum reaction rate. All experiments were performed in triplicate and the results were averaged (Li, Lu, Wu, Feng, & He, ).…”