Studies on the Interaction between Angiotensin-Converting Enzyme

Dec 4, 2018 - Institute of Chemical Sciences, University of Peshawar, Peshawar , Khyber Pakhtunkhwa 25120 , Pakistan. J. Agric. Food Chem. , Article A...
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Bioactive Constituents, Metabolites, and Functions

Studies on the interaction between angiotensin converting enzyme (ACE) and ACE inhibitory peptide from Saurida elongata Xiongdiao Lan, LiXia Sun, Yaseen Muhammad, Zefen Wang, Haibo Liu, Jianhua Sun, Liqin Zhou, Xuezhen Feng, Dankui Liao, and Shuangfei Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04303 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 4, 2018

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Journal of Agricultural and Food Chemistry

Studies on the interaction between angiotensin converting enzyme (ACE) and ACE inhibitory peptide from Saurida elongata

Xiongdiao Lan†, §, Lixia Sun‡, Yaseen Muhammad‡, II, Zefen Wang‡, Haibo Liu‡, Jianhua Sun‡, Liqin Zhou‡, Xuezhen Feng‡, Dankui Liao*‡, Shuangfei Wang*†

†College

of Light Industry and Food Engineering, Guangxi University, Nanning

530004, China ‡ School

of Chemistry and Chemical Engineering, Guangxi University, Nanning,

Guangxi 530004, China § School

of Chemistry and Chemical Engineering, Guangxi University for

Nationalities, Nanning, Guangxi 530008, China II Institute

*

of Chemical Sciences, University of Peshawar, 25120, KP, Pakistan

Corresponding authors.

Tel: +86 771 327 2702. Fax: +86 771 323 3718 E-mail address: [email protected]; [email protected]

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ABSTRACT

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Angiotensin converting enzyme (ACE) inhibitory peptides derived from food

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protein exhibited antihypertensive effects by inhibiting ACE activity. In this work, the

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interaction between ACE inhibitory peptide GMKCAF (GF-6) and ACE was studied

5

by isothermal titration calorimetry, molecular docking, UV absorption spectroscopy,

6

fluorescence spectroscopy and circular dichroism spectroscopy. Experimental results

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revealed that the binding of GF-6 to ACE was a spontaneous exothermic process

8

driven by both enthalpy and entropy. The interaction occurred via a static quenching

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mechanism and involved the alteration of the conformation of ACE. In addition, ITC

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and molecular docking results indicated binding of GF-6 to ACE via multiple binding

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sites on the protein surface. This study could be deemed helpful for the better

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understanding of the inhibitory mechanism of ACE inhibitory peptides.

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KEYWORDS: Angiotensin converting enzyme inhibitory peptides; static quenching

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interaction; molecular docking; isothermal titration calorimetry; circular dichroism

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spectroscopy

17 18 19 20 21 22 2

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INTRODUCTION

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Hypertension is a ubiquitously known cardiovascular disease which has become an

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important global public health issue. 1, 2 Angiotensin-converting enzyme (ACE) is a

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key enzyme of the renin-angiotensin and kallikrein-kinin system.

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important role in the regulation of blood pressure and cardiovascular functions.

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Inhibiting the ACE activity is regarded as a useful treatment against hypertension.

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Thus, ACE is considered as an appropriate target for the development of

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antihypertensive drugs.

3, 4

It plays an

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Since ACE inhibitor was identified in snake venom, many studies have been

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conducted for synthesizing ACE inhibitors, such as captopril, enalaprilat and lisinopril.

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Although the ACE inhibitor is the mainstay of hypertension therapy, it can produce

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substantial side effects such as cough, rash and renal dysfunction. 5-7 And this recently

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brought a greater attention for new therapeutic agents. Numerous ACE inhibitory

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peptides derived from food proteins with lesser and milder side effects have been

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developed as potent alternatives to synthesized and market-available drugs for

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preventing hypertension.

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addressed the preparation, purification and amino acid sequence analysis, while the

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direct information on the interaction of ACE inhibitory peptide and ACE was quite

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limited.

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could help us increase and improve our understanding of the influence of ACE

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inhibitory peptides in vivo and guide the suitable use of these peptides. In this

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connection, many studies on the interactions between ACE and ACE inhibitory

10-12

8, 9

Earlier studies on ACE inhibitory peptides mainly

Studying the interaction between ACE inhibitory peptides and ACE

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peptides have been reported. Fu et al.

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peptides via fluorescence and simulated their interactions usingmolecular docking.

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Xie et al.

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according to molecular docking and isothermal titration calorimetry (ITC) assay.

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These studies have provided information about the binding of ACE inhibitory

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peptides to ACE, but the mechanism has not been fully explored. Detailed and

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systematic thermodynamic, spectroscopic and computational studies on the interaction

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of peptides with ACE have not been reported in literature so far. Keeping in view the

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reputation earned by spectroscopic, calorimetric and molecular docking techniques in

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studying the protein-ligand interactions,

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investigating the interaction between ACE inhibitory peptide and ACE.

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14

analyzed the inhibition of ACE by patatin

studied the interaction mechanisms of TTW and VHW with ACE

In our previous work,

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15-17

it is worth exploring their role in

a novel ACE inhibitory peptide GMKCAF (GF-6) with

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IC50 value of 45.7 μM was isolated from lizard fish (Saurida elongate) protein

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hydrolysate. In continuation to this, the current study is focused to get insight about

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the inhibition of GF-6 on ACE via calorimetric, molecular docking and spectroscopic

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methods. ITC provided the binding constant, binding stoichiometry, enthalpy, entropy

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and free energy of GF-6-ACE interaction. Structural changes of ACE were analyzed

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by UV, fluorescence and circular dichroism (CD) spectroscopy. In addition, the mode

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of binding of peptide with ACE was simulated through molecular docking. This study

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could be beneficial for understanding the pharmacological and structural changes

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during the binding of ACE inhibitory peptides with ACE.

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MATERIALS AND METHODS 4

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Materials. ACE was prepared from pig lung according to reported literature.19,20

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Hippuryl-histidyl-leucine (HHL) as a substrate of ACE, was purchased from Sigma

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Chemical Co. (St. Louis, MO, USA). GF-6 (grade ﹥ 98%, IC50= 45.7 μM) was

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synthesized by GL Biochem (Shanghai) Ltd. (China). All the the other reagents were

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of analytical purity and used without further purification.

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Inhibition pattern of GF-6. ACE, HHL and GF-6 were dissolved in 0.1 M borate

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buffered saline (0.3 M NaCl, pH 8.3). A mixture of ACE and GF-6 was incubated in a

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water bath at 37.0 °C for 10 min. 40 μL of HHL in different concentrations (1.16 μM,

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2.32 μM, 3.49 μM, 4.66 μM, 5.24 μM and 5.82 μM) were added to the mixture. The

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reaction was carried out at 37.0 °C for 5 min and was stopped by adding 150 μL of

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1.0 M HCl. The hippuric acid liberated in the mixture was determined by reverse

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phase high pressure liquid chromatography (RP-HPLC) with an XDB-C18 (4.6×150

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mm) column. The inhibition pattern of GF-6 was determined by Lineweaver-Burk

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plot analysis, a double reciprocal plot of substrate concentration versus velocity

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according to Michaelis-Menten kinetics.

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Molecular docking. Although the crystal structure of ACE is not clear yet, human

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testicular ACE (tACE) (obtained from the Protein Data Bank (1O8A.pdb)) can fully

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replicate the function of ACE and was used as the macromolecular receptor in

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docking studies. The 3D structure of GF-6 was drawn in ChemDraw 16.0 and saved

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in .pdb format for docking studies. The Auto Dock Tools transformed the files

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from .pdb format to .pdbqt format and determined the docking box on the target.

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Molecular docking was performed using the Autodock Vina software.21 Ligand 5

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displaying the lowest binding energy in the binding pocket of protein was chosen as

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the best conformation. PyMOL was used for viewing the diagrams of protein-ligand

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interaction.

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ITC analysis. ITC measurements were performed on a Nano ITC calorimeter (TA,

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USA). ACE and GF-6 solutions were dissolved in 50 mM HEPES buffer solution (0.3

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M NaCl, pH 7.0) and properly degassed prior to titration. In a standard experiment,

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the reference cell of the calorimeter was loaded with HEPES buffer solution (0.3 M

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NaCl, pH 7.0). After stabilizing the baseline, ACE solution (2.17 μM) in the sample

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cell was titrated with GF-6 solution by 20 successive automatic injections of 2.5 μL

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each at a stirring speed of 250 rpm. The individual injections were programmed at

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intervals of 300 s, while similar experiments were performed at 15 °C, 20 °C and

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25 °C. In order to subtract the background dilution heats from the experimental data, a

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control experiment was carried out by injecting GMKCAF solution into the buffer

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alone.

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UV spectroscopic measurements. UV spectra was measured on a UV-2501PC

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Spectrophotometer (Shimadzu, Japan) attached with an externally isothermal water

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bath to maintain temperature at 15 °C, 20 °C and 25 °C. ACE and GF-6 were

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dissolved in 50 mM HEPES buffer solution (0.3 M NaCl, pH 7.0). A constant

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concentration of ACE (1.98 μM) was mixed with increasing concentration of GF-6.

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The mixture was incubated at enactment temperature for 10 min. UV absorption

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spectra were recorded in a wavelength range of 200-320 nm, which were corrected

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with respective blank having same concentration of GF-6 in buffer without ACE. 6

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Fluorescence spectroscopic measurements. Fluorescence measurements were

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performed on a LS-55 Fluorescence spectrophotometer (PE, USA). GF-6 solution was

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prepared with 50 mM HEPES buffer (0.3 M NaCl, pH 7.0) at different concentration

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and then incubated with 0.0718 μM ACE for 10 min. The emission spectra were

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recorded in wavelength range of 300-500 nm at an excitation wavelength of 285 nm.

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The excitation and emission slit widths were set at 5 nm at variable temperatures of

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15 °C, 20 °C and 25 °C using an isothermal water bath.

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CD spectroscopic measurements. CD spectral data were obtained from a

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MOS 450 CD spectrometer (Biologi, French) in rectangular quartz cuvettes of 1 mm

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path length. GF-6 solution was prepared with 50 mM HEPES buffer at different

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concentration followed by incubation with 1.12 μM ACE for 10 min. Spectra were

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recorded from 190 to 260 nm with 100 nm·min-1 scan speed and a response time of 1

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s. The background spectrum of GF-6 solution was subtracted from that of GF-6-ACE

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complex. The secondary structures of the samples were estimated from the CD spectra

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using SELCON 3 method in Dicroprot 2000 software.

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Statistical analysis. All measurements performed in triplicate and analysis of data

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variance was performed using statistical analysis system (SAS 9.1). Linear correlation

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coefficient was calculated using Origin 8.0. Values reported as significantly different

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have P values ﹤0.05.

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RESULTS AND DISCUSSION

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Inhibitory pattern of GF-6 on ACE. The inhibition kinetics of GF-6 was studied

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at different substrate concentrations, as well as varying the inhibitor level in the assay 7

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and the Lineweaver-Buck plots of the reactions are shown in Fig. 1. The lines

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intersect on 1/[s] axis indicates that GF-6 can prevent product formation though

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binding with ACE at the non-active site as a non-competitive inhibitor.

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peptides derived from food proteins such as VHW, TTW, 14 TPTQQS, 22 RYDF and

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GNGSGYVSR

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synthetic drugs (captopril, enalapril and lisinopril) are competitive inhibitors

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22

Some

have also been found to act in non-competitive pattern, while

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Molecular docking. Molecular docking of GF-6 with human tACE was carried out

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using AutoDock Vina to predict the ligand-receptor interaction mechanism. The pose

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with the lowest binding energy was recognized as the best conformation for further

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analysis.

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According to the docking results, the most stable structure of GF-6 in the

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hydrophobic cavity of tACE had a binding affinity of -7.0 kcal·mol-1, as shown in Fig.

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2a. The hydrogen bond (H-bond) could play an important role in GF-6 binding with

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tACE (Fig. 2b). The main active site of ACE is composed of three pockets i.e. S1

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pocket (Gln281, His353, Lys511, His513), S2 pocket (Ala354, Glu384, and Tyr523)

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and S1’ pocket (Glu162), which are considered as the probable contacting sites of

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competitive inhibitors with tACE. 17, 18 No interactions of GF-6 with Zn2+ is observed

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(Fig. 2b). On the other hand, GF-6 is attached to tACE primarily by the residues

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(Ser-480, Ser-481, Ala-320 and Arg-486), which were not loaded in the three active

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pockets. Protein-ligand interactions show four H-bonds with tACE. The Lys of

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peptide makes the greatest contribution to the stability of the peptide-ACE complex,

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owing to its two H-bonds with Ser-480 and Ser-481 (2.4 Å and 2.2 Å). The Cys 8

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formed one H-bond with the residues Arg-486 (2.3 Å) and Ala bound to Ala-320

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having a bond distance of 2.5 Å. These interactions kept the peptide away from the

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active site. However, previous structure-activity correlation studies have shown that

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C-terminal tripeptide of peptides plays a predominant role in binding to the active site

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of the enzyme, where position C-1 is the most relevant to the inhibitory activity. This

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disagreement may be due to the fact that these studies are based on a competitive

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inhibition mechanism in which peptides interact with the active site of ACE, while

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GF-6 (in the current study) is a non-competitive inhibitor.

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ITC assays of binding of GF-6 to ACE. The thermodynamic profiles of

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GF-6-ACE interactions obtained by ITC are shown in Fig. 3. The upper panels show

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the raw ITC profiles for the complexation of GF-6 with ACE. Each of the heat burst

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peaks corresponds to a single injection of GF-6 into the ACE solution, while the lower

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panels correspond to the corrected heats per mole of injection. The association

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constant (Ka), binding stoichiometry (n), and enthalpy change (∆H) were obtained

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after fitting of the integrated heats. The Gibbs free change (∆G) and change in entropy

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(-T∆S) were calculated using Eq. (1) and Eq. (2) respectively. 24 The thermodynamic

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parameters accompanying the binding of GMKCAF to ACE are summarized in

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Table 1.

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∆G = - RT ln(Ka)

(1)

∆G = ∆ H - T ∆ S

(2)

Where R is the general gas constant and T is the absolute temperature (K).

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Table 1 suggests a spontaneous exothermic nature of the binding process of GF-6 to

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ACE. The values of ΔG, ΔH and -T∆S decipher valuable information about the

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different binding forces involved. 25 ,26 These results further suggested that the binding

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process is concurrently driven by enthalpy and entropy. The negligible contribution of

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entropy at lower temperature implies the major role of H-bonds and electrostatic

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interactions in the process. The binding has more favorable entropy contribution at

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high temperature, suggesting that GF-6 comes in proximity to ACE through

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hydrophobic force.

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The interaction between ligand and receptor contains two types of binding: specific

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and nonspecific. “Specific binding” represents the “lock-key” interaction with strong

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affinity, while “non-specific binding” refers to the binding of non-physiological origin

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with weak affinity. 27,28 The values of association constant Ka between GF-6 and ACE

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were 4.67×105, 1.63×104 and 9.78×103 M-1 at 15 °C, 20 °C and 25 °C, respectively,

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while those reported for captopril, enalaprilat and lisinopril are 7.5×107, 1.9×108, and

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6.2×108 M-1, respectively. 29 These studies showed that all the three inhibitors bound

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with ACE at the active site via “specific binding”, while GF-6 interacted with ACE

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via “nonspecific binding”.

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Binding stoichiometry (n) values show the possibility of multiple binding sites on

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ACE for GF-6, which increased with increasing temperature. It may be due to the

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variation in ACE structure at variable temperature. Residues (exposed or buried) can

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enhance ACE interaction with GF-6. As seen from Fig. 3, saturations of binding sites

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occurred with molar ratio of approximately 67:1, 294:1 and 392:1 at 15 °C, 20 °C and 10

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25 °C, respectively. On the contrary, the saturated bindings of captopril, enalaprilat

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and lisinopril with ACE have been reported at a molar ratio of 1:1.30 The sequence of

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somatic ACE contains two potential active sites. The excessively large values of “n”

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confirm the non-active nature of these binding sites. The interaction of tannins with

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proteins (in tannin-protein binding) is primarily a surface phenomenon with higher

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number of binding sites as supported by stoichiometric studies.31-33 From ITC results,

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large number of GF-6 binding sites indicate a physiologically relevant binding and

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involves surface adsorption mechanism. A possible explanation is that ACE structure

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is flexible to transform to other conformations, and hence can provide more surface

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binding sites for non-competitive peptide. However, our molecular docking

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simulation suggested that GF-6 attached to the hydrophobic cavity of ACE. It may be

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due to the fact that docking was performed around the active site (x, 43.642; y, 38.406

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and z, 46.282) with the size of docking box as: 60 Å×60 Å×60 Å, and possible surface

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binding was not considered. To address this issue, a re-docking was performed by

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selecting the search box as large as possible to cover the entire ACE surface and the

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results are provided in Fig. 4.

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Re-docking results showed that GF-6 interacted with ACE surface with a binding

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affinity of -7.5 kcal·mol-1 whereas around active site it was -7.0 kcal·mol-1. The

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binding tends to occur on the ACE surface rather than hydrophobic cavity, which is

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confirmed from ITC assay. Fig. 4b suggests many grooves on the surface of ACE

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which equip GF-6 with large number of binding sites. In GF-6-ACE complex, peptide

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lies on the surface of ACE and makes close contacts with enzyme through H-bond 11

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formation with Thr-135, and Thr-266, Asn-249, Leu-339, and Glu-340. Where, Lys of

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peptide forms two H-bonds with Thr-135 and Thr-266, (2.1 Å and 2.2 Å); Cys forms

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three H-bonds with Asn-249, Leu-339, and Glu-340 (2.1 Å and 2.2 Å); and Ala forms

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one H-bond with Asn-249. These results suggested that Cys makes the greatest

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contribution to the stability of peptide bound with ACE.

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Recently, numerous ACE inhibitory peptides have been identified from protein

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hydrolysates, most of which are non-competitive inhibitor (bound to non-active sites)

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with much higher IC50 values than those of the competitive inhibitors. Previously, the

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inhibitory mechanisms of several ACE inhibitory peptides (competitive and

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non-competitive) were assumed to bind with ACE in the hydrophobic cavity at a ratio

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of 1:1.14,

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However, the exact location of non-active sites is unclear and hence these models may

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not be applicable. In order to predict and investigate the binding of these inhibitors,

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docking simulation should be carried out in the whole protein molecular structure.

34, 35

This binding model may explain the competitive inhibition behavior.

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Effects of GF-6 binding on the UV absorption spectra of ACE. The absorption

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spectra of ACE at different temperature shown in Fig. 5a suggest two absorption

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peaks at 226 nm and 280 nm. The strong absorption peak at 226 nm is characteristic

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of the peptide backbone, while a weaker absorption peak around 280 nm is typical of

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aromatic amino acids.36 The intensity of the peak at 226 nm dramatically changed due

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to red shifts with increasing temperature from 15 °C to 20 °C, indicating the

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disturbances to the unfolding of ACE skeleton. Further increasing temperature to

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25 °C shifts the peak at 226 nm to shorter wavelength, suggesting the folding of ACE 12

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skeleton. The intensity of the peak at 280 nm increases obviously with the increasing

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temperature indicating the exposure of buried aromatic amino acids residues.

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To further investigate the binding of GF-6 to ACE, spectral experiments were

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performed according to ITC results (Table 2). The UV absorption spectra of

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GF-6-ACE complex were obtained at constant ACE concentration while varying

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GF-6 concentration. Fig. 5b-d suggest changes in absorption spectra of ACE with the

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addition of GF-6 indicating binding interactions between the two. The intensity of

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peak at 280 nm is slightly affected with increase in GF-6 concentration, while that of

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226 nm peak shifts to longer wavelength (redshift). These results suggested that the

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binding interaction changed the hydrophobicity around the amide bonds and reflected

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the unfolding of peptide backbone. Meanwhile, the environment around aromatic

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amino acids was also affected. The different binding model might result in different

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changes of ACE under diverse conditions. Moreover, combination with a large

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number of peptides could lead to the denaturation of ACE, which provided ACE with

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a flexible pattern and hence able to change in the presence of excessive GF-6.

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Effect of GF-6 binding on fluorescence of ACE. Intrinsic fluorescence of proteins

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is mainly due to two aromatic amino acid residues, tryptophan (Trp) and tyrosine (Tyr)

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when excited at 280 nm. The fluorescence spectra of ACE at different temperature

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shown in Fig. 6a suggest a λmax of 341 nm at 15 °C, which shifted to 338 nm and 339

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nm at 20 °C and 25 °C respectively. It has been reported that residues inside the

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protein show a shorter λmax than those on or near the surface of protein. The decrease

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in λmax may be due to the movements of Trp and Tyr residues to more hydrophobic 13

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environments. The variation in λmax demonstrated a significant effect of temperature

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on the ACE structure. 37-39

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The fluorescence spectra of ACE having various concentration of GF-6 at different

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temperatures are shown in Fig. 6b-d. The fluorescence intensity of ACE decreases

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with the addition of GF-6, which is attributed to the fluorescence quenching process

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by GF-6. In order to elucidate the quenching mechanism, fluorescence data were

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analyzed according to the Stern-Volmer Equation as Eq. (3):

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F0/F=1+Kq∙τ0∙[Q]=1+KSV∙[Q]

(3)

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Where, F0 and F are fluorescence intensities of ACE in the absence and presence of

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quencher, respectively. Kq is the quenching rate constant of biomolecular reaction, τo

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is the average fluorescence lifetime of the biomolecule in the absence of quenchers,

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which is of the order of 10-8 s. KSV is the Stern-Volmer quenching constant and and

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[Q] is the concentration of the quencher.

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The Stern-Volmer plot of Fo/F versus [Q] at 25 °C is presented in Fig. 6e. The plot

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is linear before ACE saturation by GF-6, while the quenching is not regular after

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saturation. Thus, the fluorescence data before saturation at 15 °C, 20 °C, and 25 °C

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were fitted using the Stern-Volmer Equation and provided in Fig. 6f. Good linear

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relationships between F0/F and [Q] were observed, and the values of Ksv decreased

280

with increasing temperature (Table 3). In addition, the values of Kq are greater than

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the maximum value for dynamic quenching (2 × 1010 M-1·s-1). These results suggested

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that the fluorescence quenching of ACE by GF-6 was a static process and was

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initiated by the formation of a GF-6-ACE complex, not from dynamic collision. 40 14

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Conformation changes of ACE upon interaction with GF-6. Proteins with

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different structures provide different CD band positions and intensities.41,42 In Fig. 7,

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the CD spectra of ACE varied significantly with increasing concentrations of GF-6.

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For the native ACE, the contents of the secondary structural elements are of about 7.9%

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α-helices, 23.3% β-sheets, 10.3% β-turns, 9.1% P2 and 50.0% random coils. The low

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content of α-helices may be the major factor providing a flexible structure of ACE to

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contact with greater number of GF-6, while the proportion of α-helices varies greatly

291

from that found in the crystal structure of tACE. It may be attributed to the fact that

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ACE crystals were grown at 16 °C under pH 4.7, 3 while certain structure of protein

293

survives only under specific pH and temperature. As seen from Table 4, the α-helical

294

and β-sheet content of ACE decreased upon the addition of GF-6 to the ratio of 113:1,

295

revealing that GF-6 associates with the amino acid residues of ACE and destroys their

296

H-bonding networks. Upon saturation of ACE and GF-6, the contents of α-helix and

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β-sheet of ACE increase to 13.2% and 30.3%, respectively. This may be attributed to

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the larger number of GF-6 binding on ACE according to the surface adsorption

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mechanism. Compare with the saturation state, variations in the conformation of ACE

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structures are observed even at excessive amount of GF-6 than ACE. This is in

301

accordance with UV absorption spectra and fluorescence spectra results (Fig. 5b-d).

302

In summary, this study though ITC, molecular docking, UV spectroscopy,

303

fluorescence spectroscopy and CD spectroscopy, provided important quantitative data

304

for the interaction of GF-6 with ACE. Experimental results revealed that the binding

305

between GF-6 and ACE was based on complexation. GF-6 combined with ACE at 15

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non-active site as non-competitive inhibitor and effectively quenched ACE via static

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quenching mechanism. ITC and spectroscopic studies revealed that the main driven

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force changed from H-bonds to hydrophobic force with increasing temperature, which

309

was due to the flexible structure of ACE. For the GF-6-ACE complex, surface

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adsorption mechanism explained the inhibition of the non-competitive peptides with

311

higher number of binding sites, which was supported by the molecular docking results.

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This study provides important insights into the binding of GF-6 with ACE and can

313

help better understand the mechanism and mode of action of ACE inhibitory peptides.

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REFERENCES

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Funding

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Journal of Agricultural and Food Chemistry

This research was supported by the National Natural Science Foundation of China

443

(51372043),

Guangxi

Natural

Science

Foundation

444

2017GXNSFBA198215, 2017GXNSFAA198289)

23

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(2017GXNSFDA198052,

Journal of Agricultural and Food Chemistry

Figure captions: Figure 1: The Lineweave-Burk plots of the reactions of ACE in the presence of GF-6. Figure 2: The docking simulation of GF-6 binding to tACE. (a)The docking simulation of GF-6 (green) binding to tACE (shown as cartoon). A zinc ion (red) was present in the active site of tACE. (b)The interaction between GF-6 (shown as sticks) and the residues of tACE (shown as lines) is shown.

Figure 3 The heat flow of binding GF-6 to ACE (2.17 μM). (a) at 15°C,concentration of GF-6 is 1.28 mM; (a) at 20°C, concentration of GF-6 is 5.12 mM; (a) at 25°C, concentration of GF-6 is 5.12 mM Figure 4: The second molecular docking simulation of GF-6 binding to tACE. (a)The docking simulation of GF-6 (green) binding to tACE (shown as cartoon). A zinc ion (red) was present in the active site of tACE. (b) The binding of the GF-6 (green) on the surface of the ACE (orange). (c)The interaction between GF-6 (shown as sticks) and the residues of tACE (shown as lines) is shown. Red dash indicates H bonding. Figure 5: UV absorption spectra of ACE in the absence and presence of GF-6 at different concentrations. Concentration of ACE is 1.98 μM; (a) without GF-6; (b) 15°C; (c) 20°C; (d)25°C. Figure 6: Fluorescence emission spectra of ACE (0.0718 μM). (a) without GF-6; (b) 15°C; (c) 20°C; (d)25°C; (e)emission quenching curves of ACE (0.0718 μM) at 25°C; (f) emission quenching curves of ACE (0.0718 μM) at 25°C at all studied temperature Figure 7: CD spectra of ACE in the absence and presence of GF-6 at 25°C. CACE = 1.12 μM. 24

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Table 1 Thermodynamics parameters of binding between GF-6 and ACE T(°C)

n

Ka(1/M)

∆H(KJ·mol-1)

∆G(KJ·mol-1)

-T∆S(kJ·mol-1)

15

26.2

4.67×105

-31.20

-31.27

-0.07

20

103.7

1.63×104

-2.28

-23.64

-21.36

136.2

9.78×103

-3.52

-22.78

-19.26

25

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Table 2 Spectroscopic experimental conditions T(°C)

GF-6:ACE

Titration number of ITC

15

0:1

-

28:1

3

67:1

7

119:1

12

212:1

20

0:1

-

113:1

3

294:1

7

611:1

15

849:1

20

0:1

-

113:1

3

392:1

10

611:1

15

849:1

20

Saturation

20

Saturation

25

Saturation

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Table 3 Stern-Volmer quenching constants (KSV and Kq) due to binding between GF-6 and ACE

T (°C) 25 20 15

104

Ksv L/mol

1012

0.94 1.51 5.37

Kq L/mol·S 0.94 1.51 5.37

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R 0.9989 0.9933 0.9135

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Table 4 Secondary structures of ACE affected by GF-6 at 25 °C GMKCAF:ACE

α-helices % (±0.1)

β-sheetss % (±0.2)

β-turns % (±0.1)

P2 % (±0.1)

Random coils % (±0.3)

0:1

7.9

23.3

10.3

9.0

50.0

113:1

5.5

22.8

10.7

9.0

51.0

292:1

13.2

30.3

9.1

9.7

28.0

611:1

11.9

28.6

8.2

9.3

29.0

848:1

12.1

33.6

9.3

10.5

32.0

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Fig. 1

control 30.4M GF-6 60.8M GF-6

1000

-1

1/v(M min)

800

-2

-1

600 400 200 0 -200

0

1

2

3

4

5

-1

1/[S](mM )

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Fig. 2

a

b

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Fig. 3

a

b

c

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Fig. 4

a

b

c

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Fig. 5

a

1.2

b

15C 20C 25C

1.0

GF-6ACE=01 GF-6ACE=281 GF-6ACE=671 GF-6ACE=1191 GF-6ACE=2121

1.2 1.0 0.8 0.6

0.8

0.4 0.2

A

A

0.6 0.4

0.0 -0.2 -0.4

0.2

-0.6 -0.8

0.0

-1.0

220

240

260

280

300

220

320

240

260

c

1.2

0.8 0.6

300

320

1.2 GF-6ACE=01 GF-6ACE=1131 GF-6ACE=3921 GF-6ACE=6111 GF-6ACE=8491

1.0 0.8 0.6

0.4

0.4

0.2

0.2 A

A

d

GF-6ACE=01 GF-6ACE=1131 GF-6ACE=2941 GF-6ACE=6111 GF-6ACE=8491

1.0

280 nm

nm

0.0

0.0

-0.2

-0.2

-0.4

-0.4

-0.6

-0.6

-0.8

-0.8 -1.0

-1.0 220

240

260

280

300

220

320

240

260 nm

nm

33

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300

320

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Fig. 6

a

300 250

Flourescence Intensity

b

15C 20C 25C

300 GF-6ACE=01 GF-6ACE=281 GF-6ACE=671 GF-6ACE=1191 GF-6ACE=2121

250

200

200

150

F

150

100

100

50

50 0 300

0 300

320

340

360

380

400

420

440

460

480

500

320

340

360

380

400

c

d

300 GF-6ACE=01 GF-6ACE=1131 GF-6ACE=2941 GF-6ACE=6111 GF-6ACE=8491

250 200 F

100

50

50

360

380

400

420

440

460

480

0 300

500

320

340

360

380

400

f

1.20

1.16

1.15

1.12

1.10

1.04

1.00

1.00 10

15

20

440

460

480

1.08

1.05

5

420

25

30

35

15 C 20 C 25 C

1.20

F/F0

F/F0

1.25

0

500

nm

nm

e

480

150

100

340

460

GF-6ACE=01 GF-6ACE=1131 GF-6ACE=3921 GF-6ACE=6111 GF-6ACE=8491

200 F

320

440

300 250

150

0 300

420

nm

Wavelength(nm)

-2

[Q]

0

2

4

6

8 [Q]

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12

14

16

18

500

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Fig. 7

14000

GF-6:ACE=0:1 GF-6:ACE=113:1 GF-6:ACE=392:1 GF-6:ACE=611:1 GF-6:ACE=849:1

12000 10000

Ellipticities

8000 6000 4000 2000 0 -2000 -4000 -6000 -8000 190

200

210

220

230

240

250

260

Wavelength(nm)

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Table of Contents Graphic (TOC)

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