Inhibitory mechanism of a substrate-type angiotensin I-converting enzyme inhibitory peptide

Accepted Manuscript Title: Inhibitory mechanism of a substrate-type angiotensin I-converting enzyme inhibitory peptide Authors: Junjie Wu, Dewei Xie, Xujun Chen, Ya-Jie Tang, Lixin Wang, Jingli Xie, Dongzhi Wei PII: DOI: Reference:

S1359-5113(18)31439-9 https://doi.org/10.1016/j.procbio.2018.12.018 PRBI 11529

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

18 September 2018 17 November 2018 18 December 2018

Please cite this article as: Wu J, Xie D, Chen X, Tang Y-Jie, Wang L, Xie J, Wei D, Inhibitory mechanism of a substrate-type angiotensin I-converting enzyme inhibitory peptide, Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.12.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inhibitory mechanism of a substrate-type angiotensin Iconverting enzyme inhibitory peptide

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Junjie Wua, Dewei Xiea, Xujun Chena, Ya-Jie Tangb, Lixin Wangc, Jingli

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Xiea,d,*, Dongzhi Weia,d,*

State Key Laboratory of Bioreactor Engineering; Department of Food Science and

Technology, School of Biotechnology, East China University of Science and

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b

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Technology, Shanghai 200237, P. R. China

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Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei

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Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei Key

430068, P. R. China

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Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan

Sports Health Center. Tongji University, Shanghai 200092, P. R. China

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Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB),

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c

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Shanghai 200237, P. R. China * Corresponding author. Address: P. O. Box 283, East China University of Science

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and Technology, 130 # Meilong Rd, Shanghai 200237, P. R. China. Fax: +86-2164252563. E-mail address: [email protected] (J. Xie), [email protected] (Wei D).

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Graphical abstract

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ITC assay indicated ACE inhibitory peptide GNGSGYVSR may be hydrolyzed by

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Highlight

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ACE

HPLC analysis imply GNGSGYVSR as a substrate-type inhibitor of ACE

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Hydrolysate GNGSGYV and SR was identified by UPLC & Q-TOF MS

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Inhibitory mechanism of GNGSGYVSR as substrate-type inhibitor of ACE was

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discussed

ABSTRACT

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The inhibitory mechanism during the reaction between peptides from Phascolosoma esculenta and angiotensin I converting enzyme (ACE) was studied. Thermodynamics features of the peptide-ACE binding reaction measured by isothermal titration calorimetry (ITC) assay, combining with the decrease of ACE inhibition within 24 h implied that Gly-Asn-Gly-Ser-Gly-Tyr-Val-Ser-Arg decomposed during the reaction

and played as a substrate-type inhibitor. The hydrolysate Gly-Asn-Gly-Ser-Gly-TyrVal (GNGSGYV) without ACE inhibitory activity was identified by UPLC & Q-TOF MS, while the hydrolysate Ser-Arg (SR) showed competitive inhibition of ACE with IC50 value of 790 μM. However, GNGSGYV and SR have synergistic effect on ACE

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and result in an ACE inhibitory IC50 value of 170 μM. The synergistic mechanism illustrated by two-step molecular docking discovered that SR firstly attacked the

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catalytic Zn of ACE and formed coordinate bond, and then GNGSGYV bound with the arginine of SR and residues along the channel of ACE active site by hydrogen

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bonds to block the substrate from entering. The circular dichroism spectra also

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verified that SR and GNGSGYV significantly changed the secondary structure of

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ACE.

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Keywords: substrate-type ACE inhibitory peptide, inhibitory mechanism, ITC,

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molecular docking

1. Introduction Hypertension is a major health concern afflicting approximately a quarter of adults worldwide. The affected population is predicted more than 1.5 billion by 2025 [1]. Blood pressures were regulated through multiple mechanisms, and one was

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demonstrated that blood pressure was elevated by angiotensin I converting enzyme (ACE, EC 3.4.15.1) [2]. Therefore, inhibitors of ACE are the first line of therapy for

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hypertension. Except drugs such as lisinopril, captopril and enalapril for the treatment of hypertension, food-derived ACE inhibitory peptides become notable because that

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their natural origin goes along with the raising demands of controlling blood pressure

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in a safer way. Up to now, ACE inhibitory peptides have been discovered in various

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kinds of food, such as dairy products [3-5], seafood [6-8] and plants [9-11].

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Most works concerning food borne ACE inhibitory peptides reported the discovery

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of ACE inhibitory peptides, activity assay as well as the illustration of inhibitory mechanism. With the development of bioinformatics, structural biology and

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computational biology, method in silico such as molecular docking was widely used

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in both virtual screening of ACE inhibitory peptides and inhibitory mechanism revealing in recent years [12,13]. There were some docking software for such purpose were reported, such as Autodock [14], Bindsurf [15], Metadock [16], LeadFinder

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[17,18], Vina [19,20], FlexScreen [21,22], and Blind docking server (http://biohpc.ucam.edu/achilles/). Autodock is an open source of molecular docking software, which is applied in many works on ACE inhibitory peptides [14,23-25]. More recently, isothermal titration calorimetry (ITC) assay, previously used in drug

research, was found to be an efficient tool in the illustration of the binding between peptides and ACE based on the thermodynamic characteristics determined [26]. After pre-incubated with ACE, inhibitory peptides can be classified into three groups according to the variation of inhibitory reaction: (1) inhibitor-type with

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invariable ACE inhibition; (2) prodrug-type with increased inhibition and (3) substrate-type with decreased inhibition [27]. Inhibitor-type peptides anchor with

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ACE without further hydrolysis. On the contrary, inhibitory peptides of both prodrugtype and substrate-type can be cleaved by ACE during the incubation, followed by

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releasing new ACE inhibitor or losing activity. Thus, the role of inhibitors playing

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during the reaction phase must be considered because that the inhibitory activity

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fluctuation within a certain period is essential information for the application purpose.

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However, current experimental and computational methods mainly referred to the

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initial stage of peptide binding, rather than the intrinsic change of peptides during interaction with ACE [26,28,29]. For instance, three novel ACE inhibitory

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oligopeptides, Arg-Tyr-Asp-Phe (RYDF), Tyr-Ala-Ser-Gly-Arg (YASGR) and Gly-

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Asn-Gly-Ser-Gly-Tyr-Val-Ser-Arg (GNGSGYVSR), purified from the gastrointestinal hydrolysate of Phascolosoma esculenta in our previous study. The inhibitory mechanism of such peptides was surveyed through molecular docking

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illustrating the binding between original peptides and ACE, which did not take into account of the possible change of the peptides during the reaction with ACE. Nevertheless, a prospective ACE inhibitory peptide should maintain its ACE inhibition for a longer period than, for example, 1 h or even longer, which is

significant for its usage in blood pressure control in vivo. Accordingly, the reaction between peptide and ACE for a duration is necessary observed to understand the real role of peptide and its prospect in pharmaceutic application. Therefore, the aim of present work is to discover the inhibitory mechanism during

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the reaction instead of the immediately binding. Firstly, thermodynamic parameters of above peptides binding to ACE were determined through ITC assay. Then, the

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inhibitor property of such peptides against ACE was assessed by the measurement of peptides stability versus ACE. In final, the detailed inhibitory mechanism during the

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incubation of peptide and ACE was illustrated via molecular docking, and the binding

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2.1. Materials

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2. Materials and methods

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site and steric effect of inhibitory peptides were also discussed.

ACE from rabbit lung (≥ 2.0 U/mg protein) and hippuryl-histidyl-leucine (HHL) as

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a substrate of ACE were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

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Acetonitrile and trifluoroacetic acid (TFA) were purchased from Aladdin (Shanghai, China). All other reagents used in this study were analytical grade chemicals.

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2.2. Peptide synthesis All peptides, were synthesized by the Synpeptide Co., Ltd. (Shanghai, China). The

purity (≥ 95%) of the peptides was certified by HPLC column (Inertsil ODS-SP: 4.6 mm × 250 mm × 5 μm, Shimadzu Co. Ltd., Kyoto, Japan). 2.3. Isothermal titration calorimetry (ITC) binding assays

Assays were performed using an iTC200 microcalorimeter (GE, Fairfield, CT, USA) according to our previous report [26]. Briefly, all samples were ACE consisted of 0.1 M sodium borate buffer (pH 8.3), which was titrated by inhibitory peptides or lisinopril at 25 C. For the control, H2O was used instead of peptide. The ACE

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concentration was maintained at 80 to 140 nM, which was one-tenth of the peptide concentration. Each titration consisted of 18 times of 2-μL injections of the peptide

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into ACE solution. The reference power and stirring speed were 5 μcal/s and 1000 rpm respectively. Thermodynamic parameters (G, H, and S) and dissociation

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constant Kd of the peptide-ACE interaction were determined.

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2.4. Measurement of ACE inhibition

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ACE inhibition was measured in vitro following the spectrophotometric assay

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described by Guo et al. with some modification [30]. An improved linear gradient was

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conducted by acetonitrile (20 to 10%, 0-10 min; 10%, 10-16min). Triplicate tests were performed for each sample. IC50 value was defined as the concentration of the peptide

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inhibiting 50% of the ACE activity.

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2.5. Stability of the peptides to ACE The stability of the three peptides against ACE was according to the method of our

previously reported work [26]. Briefly, 25 μL peptide solution (0.2 mg/mL) mixed

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with 10 μL of ACE solution (310 mU/mL) was incubated at 37 °C for various periods. The ACE inhibition of the mixture was measured as description of section 2.4 in different interval such as 0.5 h, 1 h, 2 h, 4 h and 8 h during the whole reaction period. 2.6. Peptide sequence analysis by UPLC & Q-TOF MS

The hydrolyzed product of GNGSGYVSR was applied to ACQUITY UPLC I-Class system & VION IMS Q/TOF Mass spectrometer (Waters Corp., Milford, MA, USA) with UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm, Waters). The mobile phase flew at a rate of 0.4 mL/min with a linear gradient conducted by eluent B (5 to 100 %

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during 40 min). The scan mode including two independent scans with different collision energies (CE) were alternatively acquired during the run to fragment the

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ions. Argon (99.999%) was used as collision-induced dissociation gas. The scanning range was between 50 and 1000 Da. Data were acquired by software UNIFI 1.8.1

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(Waters Corp., Milford, MA, USA).

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2.7. Determination of inhibitory pattern

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Twenty-five microliter peptide solution (1 mg/mL and 0.5 mg/mL) were added to

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reaction mixture with different concentrations of HHL (5.8 mM, 2.9 mM, 1.45 mM

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and 0.725 mM). ACE inhibitory activity was determined and then the kinetics of the reaction was deduced by Lineweaver-Burk plots.

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2.8. Molecular docking

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The crystal structure of human testicular ACE (PDB ID: 1O8A), which was widely used as receptor in docking experiment [28,29,31], was obtained from the protein data bank (http://www.pdb.org) for the docking receptor. The structure of peptides was

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constructed with ChemBio Office and the energy minimized under CFF (consistent force-field, class II all-atom force-field, especially for the molecules with smaller weight such as peptides [32,33]) module of Discovery Studio 4.0 software (San Diego, CA, USA). Prior to the molecular docking, water molecules and the inhibitor

lisinopril in 1O8A were removed using DS 4.0, while the cofactors zinc and chloride ions were retained. Autodock Tools 4.2 was used to carry out the molecular docking with the Lamarckian genetic algorithm. Since the zinc ion was an important catalytic component of ACE and the residues of ACE close to zinc ion played a vital role for

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the catalysis [2], the coordinate of zinc (x: 43.817, y: 38.308 and z: 46.652) was selected as the center of the grid box. The box size was 65 Å × 65 Å × 65 Å points for

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the peptide according to the volume of the biggest ligand GNGSGYVSR (1259.8 Å3) for the ligand freely twisting inside the box. Grid spacing of 0.375 was chosen for

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balancing accuracy and efficiency of the docking. The best docking poses of the

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peptides among 100 ga_runs were obtained according to the binding-energy value

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(G). All the docking diagrams were performed using DS 4.0.

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2.9. Chirascan circular spectroscopy measurements

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Chirascan circular (CD) spectral data were obtained from a CD Dichroism spectrometer (Applied photophysics Ltd., Britain) in rectangular quartz cuvettes of 1

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mm path length. Different peptide was dissolved in ultrapure water, then peptide

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solution was incubated with 1.2 M ACE for 10 min at 37 °C, and the molar ratio of ACE: peptide was set as 1:100. Spectra was recorded from 190 to 260 nm with 120 nm/min scan speed. The secondary structure contents of the samples were estimated

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from the CD spectra using CDNN software. 2.10. Statistical analysis All assays were conducted in triplicate, with data expressed as means ± standard errors. Statistical treatment of data was performed by one-way ANOVA with Minitab

v17 (Minitab Ltd., UK).

3. Results and discussion 3.1. ITC Assays

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The thermodynamic parameters, the binding free energy (G), binding enthalpy (H) and entropy (S) of the reactions between three peptides and ACE determined

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by ITC with Lisinopril as the positive control were shown in Fig. 1 and Table 1.

Lisinopril showed a rapid reaction with ACE according to Fig. 1a. Besides, the lowest

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dissociation constant Kd of the inhibitor-ACE complex characterizing the binding

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affinity indicated that Lisinopril-ACE is the most stable complex, which results in

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total loss of the ACE activity. GNGSGYVSR with the highest inhibitory activity (IC50

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= 25 M) exhibits the lowest G, -13.5 kcal/mol, while YASGR (IC50 = 184 M) and

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RYDF (IC50 = 235 M) show the higher G, -11.3 kcal/mol and -6.0 kcal/mol, respectively. Based on the values of H and -TS, G values of the reactions of both

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GNGSGYVSR and YASGR are entropically contributed. The values of -TS indicate

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the degree of ACE conformational variations during the reaction [34], suggesting the sufficient interactions between ACE and GNGSGYVSR or YASGR. GNGSGYVSRACE complex also exhibited lower Kd, meanwhile those of the complexes YASGR-

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ACE and RYDF-ACE show approximate dozens of times and 100,000 times high, verifying that the nonapeptide can easily bind with ACE and forms more stable peptide-ACE complex than the rest two during this reaction period. It is interesting that the ITC profile of GNGSGYVSR changes at 18 min, which indicates that the

stability of complex GNGSGYVSR-ACE may be destructed due to the decomposition of GNGSGYVSR. Accordingly, the stability of the peptides for prolonged duration needs to be discovered. 3.2. Peptide stability against ACE

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The stability of three peptides on ACE was investigated depending on the inhibitory activity after different periods of ACE incubation. As shown in Fig. 2, the ACE

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inhibitory activities of RYDF and YASGR are constant during 24 h of reaction with ACE. On the contrary, ACE inhibitory activity of GNGSGYVSR begin to decrease

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even at the first 30 min of the reaction, and left 23% at 12 h of incubation, and then

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the inhibitory activity maintained steady at such level until 24 h. The HPLC analysis

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of the reaction mixture at 4 h, further revealed that both RYDF and YASGR were

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stable against ACE (Fig. 3a, b), but a new compound formed in the mixture of

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nonapeptide (Fig. 3c). C-terminal dipeptide SR was inferred to be removed by ACE and heptapeptide GNGSGYV was released according to the sequence identification of

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the heptapeptide by UPLC & Q-TOF MS (Fig. 4), despite the dipeptide hydrolysate

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was not examined due to its lower hydrophobicity. The observation agreed with the catalytic property of ACE that ACE could cleave C-terminal dipeptide or tripeptide amide from its substrate [35]. The time-course of GNGSGYVSR converting to

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GNGSGYV during 12 h (Fig. 5) shows that the peak of GNGSGYVSR gradually shortens while the peak of GNGSGYV rises. The result interprets that ACE inhibitory activity decline of GNGSGYVSR is due to its hydrolysis by ACE. It has been reported that ACE acts on a diverse range of substrates and displays

both exopeptidase and endopeptidase activity. According to the substrate selectivity and cleavage site of ACE on several reported oligopeptides [27,36,37], cyclical and aromatic amino acids were found in majority of P1 and P1’ sites of the peptides, such as proline, phenylalanine, tyrosine and histidine. Moreover, ACE preferred substrates

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with cyclical and aromatic amino acids at P1 site in spite of its wide range of substrate specificity. Most of the oligopeptide substrates were removed a dipeptide from C-

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terminus by ACE, and a few tripeptide products instead. ACE exhibits dipeptidase

activity on most substrates rather than tripeptidase or endopeptidase activity. In the

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present work, ACE showed dipeptidase activity to GNGSGYVSR, and the

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penultimate Val-Ser bond was cleaved. Since both valine and serine are hydrophobic

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amino acids without cyclical and aromatic groups, the cleavage mode is obviously

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different from most reported oligopeptide substrates with cyclical and aromatic amino

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acids at P1 or P1’ site. FFGRCVSP, an octapeptide from ovalbumin [27], shared the same cleavage site as GNGSGYVSR.

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3.3. ACE inhibitory activity and inhibition pattern of the produced peptides

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ACE inhibitory activities of synthesized SR and GNGSGYV were determined (Table 2). The IC50 value of SR was 790 μM, however GNGSGYV did not show potent inhibitory activity even at the concentration of 2 mg/mL. These results

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suggested that GNGSGVYSR is the substrate-type inhibitor of ACE, hydrolyzed into SR and GNGSGYV with higher IC50. Both RYDF and YASGR are confirmed as true inhibitor with constant IC50. The dipeptide exhibits much less inhibitory activity than the nonapeptide in accordance with the time course of the ACE inhibitory activity of

GNGSGYVSR. According to the results showed in Fig. 2 and Fig. 5, SR and GNGSGYV began to play roles on ACE inhibition when most GNGSGYVSR was hydrolyzed. Therefore, synergistic effect of SR and GNGSGYV on ACE inhibitory activity was determined. The IC50 value of the mixture of SR and GNGSGYV was

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170 μM, lower than that of SR alone. The result gave a clue that the presence of both peptides made more space of the ACE active pocket be occupied and led to the

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inhibition increasing.

Inhibition pattern of SR was also determined by the Lineweaver-Burk plots (Fig.

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6). The deduced kinetics parameters were shown in Table 4. The constant Vmax and

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decreased Km indicated that SR acted as a competitive ACE inhibitor. The dipeptide

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competes for binding to the ACE active sites, and then blocked ACE from interacting

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with substrate. The ACE inhibitory activity was more significant when the amount of

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SR was increased.

The Km of ACE substrate HHL on uninhibited ACE is 6.49 mM, while this value

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dose-dependently decreases in the presence of SR. It supposed that the peptide

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occupied the active site of ACE, and formed ACE-SR complex. This complex prevented HHL from binding to ACE and subsequently decreased the affinity between ACE and HHL. Higher Ki value correlated with the lower ACE inhibitive activity,

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therefore, SR with the lower ACE inhibitory activity showed the higher Ki value, 2736 mM than that of GNGSGYVSR, 297 mM. 3.4. Inhibitory mechanism based on molecular docking The binding free energy (G) of the combination, which could well indicate the

binding affinities between the ligand and receptor in negative correlation [38], was predicted through Autodock Tools 4.2 and shown in Table 2 with IC50 values. The values of G and IC50 are well consistent in all the case of GNGSGYVSR and its products of ACE hydrolysis. GNGSGYVSR has the lowest G value of -13.3 kJ/mol,

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but when it loses C-terminal SR by the hydrolysis of ACE, the binding between the peptide and ACE is weakened and results in the highest G. The ACE inhibitory of

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the produced GNGSGYV almost disappears. Nevertheless, the synergistic binding of

SR and GNGSGYV with ACE shows different result from the single peptide reaction.

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Moreover, the binding order also has the effects on the predicted G, when the two

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peptides dock to 1O8A-SR or 1O8A-GNGSGYV complex respectively. Under the

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situation that SR binds with ACE firstly, the G value of the synergistic binding was

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coincided with IC50 values.

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lower, implied the complex 1O8A-SR-GNGSGYV is more stable, which is well

Details of interaction between ACE and peptides were shown in Fig. 7.

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GNGSGYVSR forms the most hydrogen bonds with ACE residues (Fig. 7a).

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GNGSGYVSR is stabilized by plentiful hydrogen bonds with residues within the neighborhood of the entrance channel of ACE active pocket instead of the active pocket of ACE illustrated by the docking using Autodock Tools 4.2, thus the peptide

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can’t form interaction with Zn (II). Because the conformation of ACE in Autodock Tools 4.2 is rigid, the docking results was different from the results obtained using Flexible Docking module of DS 3.5 [30], due to the computational principal of Flexible Docking depending on the conformation flexibility of ACE. Moreover, the

pose of GNGSGYVSR within the peptide-ACE complex illustrated by Autodock Tools 4.2 indicates its noncompetitive property. There are four hydrogen bonds formed between Arg522 of ACE and the peptide. Arg522 was reported as a significant ligand to the second Cl¯ of ACE and the binding site for lisinopril and the substrate of

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ACE as well [2], which may contribute to the potent inhibitory activity. Although the amount of hydrogen bonds between SR and ACE is the least among

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the three peptides, the inhibitory activity of SR is much higher than GNGSGYV may due to a coordination bond formed between SR and Zn (II) (Fig. 7b). Zn (II) at the

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ACE active site usually plays a significant role for ACE activity, which constitutes a

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tetrahedrally-coordinated Zn (II) with His383, His387, Glu411 of ACE [2]. Thus, this

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combination prevented the substrate from binding with the cation and increased the

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ACE inhibitory activity. ACE has three main active site pockets (S1, S2 and S1’). S1

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pocket contains Ala354, Glu384 and Tyr523 residues, S2 pocket contains Gln281, His353, Lys511, His513 and Tyr520 residues, and S1’ includes Glu162 residue [2].

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Four hydrogen bonds were formed between Ser residue of the peptide and S1 pocket,

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and three hydrogen bonds between Arg and S1 pocket. As shown in Fig. 8a, SR bonds with Zn (II) and S1 pocket tightly in accord with its competitive inhibition mode. Electrostatic force between SR and Arg522 instead of hydrogen bond might also

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partly interpret the significant distinction between the two IC50 values of SR and GNGSGYVSR. The C-terminal GYV of GNGSGYV stretched into active pocket, and main interactions happened in S2 pocket. The N-terminal Gly was completely out of the

pocket, and combined with Glu411 and Pro407 for the stability of the peptide-ACE complex (Fig. 7c). Besides, GNGSGYV fails to build coordination with both Zn (II) and electrostatic force with Arg522 resulting its extreme low ACE inhibitory activity. The shortest distance between neighboring oxygen atom and Zinc atom was 2.55 Å,

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which was further than average length of Zn-O coordination bond (~1.95 Å) [39]. Therefore, it is difficult for the formation of the interaction between oxygen atom and

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Zinc.

3.5. Synergistic effect of SR and GNGSGYV on ACE inhibition

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Three N-terminal helices of ACE, α1 (Asp40-Thr71), α2 (Thr74-Leu107) and α3

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(Asn109-Gln120), composes a ‘lid’ on the top of the active-site cleft (Fig. 8a).

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Substrate seems to enter the catalytic pocket through the lid, which can also prevent

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large polypeptide from hydrolysis by ACE. GNGSGYVSR blocks the entrance of the

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active pocket of ACE relied on its steric occupation (1259.8 Å3) and stable interaction with the residues within the hydrophobic cavity outside of the active site of ACE, and

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then resulted the potent ACE inhibition. On the contrary, SR anchors to the deep

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active site of ACE however it smaller steric occupation makes the channel spacious for substrate binding. According to the G values shown in Table 2, the reaction after 12 h of ACE with

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GNGSGYVSR can be illustrated as two-step process that firstly SR binds with ACE, and then GNGSGYV binds with ACE-SR complex (Fig. 8b). GNGSGYV interacts with the residues along the channel, and its location was fixed by three hydrogen bonds with the Arg of SR. The steric volume (1052.0 Å3) of GNGSGYV partially

hindered the substrate entrance and improved the total ACE inhibitory activity. The ACE inhibition mechanism of SR and GNGSGYV mixture can be summarized as that SR attacks the catalytic Zn of ACE, and formed coordinate bond in the first-step reaction. Then GNGSGYV binds with the arginine of SR and residues along the

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channel by 10 hydrogen bonds to block the substrate from entering the active site pocket.

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The secondary structural information of ACE and four peptide-ACE complexes is indicated by CD spectra (Fig. 9.). The effects of peptide binding on the secondary

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structure of ACE are notable for all peptides. When SR and GNGSGYV together bond

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with ACE, the change of ACE secondary structure is the most significant. The results

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verify that SR and GNGSGYV have synergistic influence on ACE conformation.

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4. Conclusion

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ACE inhibitory peptide GNGSGYVSR can release C-terminal dipeptide SR, a competitive inhibitor of ACE, by ACE hydrolysis. The result indicates that

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GNGSGYVSR is a substrate-type inhibitor. SR and GNGSGYV exhibit significant

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decreased ACE inhibition compared with the precursor, however, these peptides show the synergistic effect on ACE inhibition, and the activity are higher than that of the sole peptide. The inhibitory mechanism based on molecular docking and two-step

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docking discovered that the precursor peptide and two products displayed different steric occupations to the active pocket of ACE, CD spectra also evidenced the synergistic roles of the two hydrolysates. Conflicts of interest

The authors declare no competing financial interest. Acknowledgements This work was supported by the Open Funding Project of Key Laboratory of Fermentation Engineering (Ministry of Education) of China, and the Opening Project

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of Shanghai Key Laboratory of New Drug Design (Grant No. 17DZ2271000), China.

References [1] P. Y. Courand, P. Lantelme, Stretching the carotid sinus to treat resistant hypertension. Lancet 390 (2017) 2610-2612. [2] R. Natesh, S. Schwager, E. D. Sturrock, K. R. Acharya, Crystal structure of human

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angiotensin converting enzyme-lislinopril complex. Nature 421 (2003) 551-554. [3] H. R. Ibrahim, A. S. Ahmed, T. Miyata, Novel angiotensin-converting enzyme

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inhibitory peptides from caseins and whey proteins of goat milk. J. Adv. Res. 8 (2017) 63-71.

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[4] Y. Li, F. A. Sadiq, T. J. Liu, J. C. Chen, G. Q. He, Purification and identification of

N

novel peptides with inhibitory effect against angiotensin I-converting enzyme and

A

optimization of process conditions in milk fermented with the yeast Kluyveromyces

M

marxianus. J. Funct. Foods 16 (2015) 278-288.

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[5] M. A. Corrons, C. S. Liggieri, S. A. Trejo, M. A. Bruno, ACE-inhibitory peptides from bovine caseins released with peptidases from Maclura pomifera latex. Food Res.

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Int. 93 (2017) 8-15.

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[6] S. W. A. Himaya , D. H. Ngo, B. M. Ryu, S. K. Kim, An active peptide purified from gastrointestinal enzyme hydrolysate of Pacific cod skin gelatin attenuates angiotensin-I converting enzyme (ACE) activity and cellular oxidative stress. Food

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Chem. 132 (2012) 1872-1882. [7] B. Forghani , M. Zarei, A. Ebrahimpour, R. Philip, J. Bakar, A. A. Hamid, Saari N. Purification and characterization of angiotensin converting enzyme-inhibitory

peptides derived from Stichopus horrens: Stability study against the ACE and inhibition kinetics. J. Funct. Foods 20 (2016) 276-290. [8] W. Ji, C. Zhang, H. Ji, Two novel bioactive peptides from Antarctic krill with dual angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory activities. J.

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Food Sci. 82 (2017) 1742-1749. [9] A. Moayedi, L.Mora, M. C. Aristoy, M. Safari, M. Hashemi, F. Toldrá, Peptidomic

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analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteins fermented using Bacillus subtilis. Food Chem. 250 (2018) 180-187.

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[10] P. Zhang, S. Roytrakul, M. Sutheerawattananonda, Production and purification of

N

glucosamine and angiotensin-I converting enzyme (ACE) inhibitory peptides from

A

mushroom hydrolysates. J. Funct. Foods 36 (2017) 72-83.

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[11] L. Paiva, E. Lima, A. I. Neto, J. Baptista, Isolation and characterization of

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angiotensin I-converting enzyme (ACE) inhibitory peptides from Ulva rigida, C. Agardh protein hydrolysate. J. Funct. Foods 26 (2016) 65-76.

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[12] H. Wu, Y. Liu, M. Guo, J. Xie, X. M. Jiang, A virtual screening method for

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inhibitory peptides of angiotensin I-converting enzyme. J. Food Sci. 79 (2014) 16351642.

[13] Z. Yu, S. Wu, W. Zhao, L. Ding, D. Shiuan, F. Chen, J. Li, J. Liu, Identification

A

and the molecular mechanism of a novel myosin-derived ACE inhibitory peptide. Food Funct. 9 (2017) 364-370.

[14] C. Liu, L. Fang, W. Min, J. Liu, H. Li, Exploration of the molecular interactions between angiotensin-I-converting enzyme (ACE) and the inhibitory peptides derived from hazelnut (Corylus heterophylla Fisch.). Food Chem. 245 (2018) 471-480. [15] I. Sánchez-Linares, H. Pérez-Sánchez, J. M. Cecilia, J. M. García, High-

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throughput parallel blind virtual screening using bindsurf. BMC Bioinformatics 13 (2012) 1-14.

SC R

[16] B. Imbernón, J. M. Cecilia, H. Pérez-Sánchez, D. Giménez, Metadock: a parallel

metaheuristic schema for virtual screening methods. Int. J. High Perform. C. 4 (2017)

U

1-15.

N

[17] J. P. Cerón-Carrasco, J. Cerezo, A. Requena, J. Zuñiga, J. Contreras-García, S.

A

Chavan, M. Manrubia-Cobo, H. Pérez-Sánchez, Labelling Herceptin with a novel

ED

J. Mol. Model. 20 (2014) 2401.

M

oxaliplatin derivative: a computational approach towards the selective drug delivery.

[18] C. M. Martínez-Ballesta, H. Pérez-Sánchez, D. A. Moreno, M. Carvajal, Plant

PT

plasma membrane aquaporins in natural vesicles as potential stabilizers and carriers of

CC E

glucosinolates. Colloid. Surface. B. 143 (2016) 318-326. [19] J. P. Cerón-Carrasco, H. Den-Haan, J. Peña-García, J. Contreras-García, PérezSánchez, H. Exploiting the cyclodextrins ability for antioxidants encapsulation: a

A

computational approach to carnosol and carnosic acid embedding. Comput. Theor. Chem. 1077 (2016) 65-73. [20] G. Budryn, B. Pałecz, D. Rachwał-Rosiak, J. Oracz, D. Zaczyńska, S. Belica, I. Navarro-González, J. M. V. Meseguer, H. Pérez-Sánchez, Effect of inclusion of

hydroxycinnamic and chlorogenic acids from green coffee bean in β-cyclodextrin on their interactions with whey, egg white and soy protein isolates. Food Chem. 168 (2015) 276-287. [21] J. Navarro-Fernández, H. Pérez-Sánchez, I. Martínez-Martínez, I. Meliciani, J. A.

IP T

Guerrero, V. Vicente, J. Corral, W. Wenzel, In silico discovery of a compound with nanomolar affinity to antithrombin causing partial activation and increased heparin

SC R

affinity. J. Med. Chem. 55 (2012) 6403–6412.

[22] H. Pérez-Sánchez, V. Rezaei, V. Mezhuyev, D. Man, J. Peña-García, H. den-

U

Haan, S. Gesing, Developing science gateways for drug discovery in a grid

N

environment. SpringerPlus 5 (2016) 1300.

A

[23] G. Vistoli, A. Pedretti, A. Mazzolari, B. Testa, Homology modeling and

M

metabolism prediction of human carboxylesterase-2 using docking analyses by

(2010) 771-787.

ED

GriDock: a parallelized tool based on AutoDock 4.0. J. Comput. Aid. Mol. Des. 24

PT

[24] R. Norris , F. Casey, R. J. FitzGerald, D. Shields, C. Mooney, Predictive

CC E

modelling of angiotensin converting enzyme inhibitory dipeptides. Food Chem. 133 (2012) 1349-1354. [25] N. S. H. N. Moorthy, N. F. Bras, M. J. Ramos, P. A. Fernandes, Binding mode

A

prediction and identification of new lead compounds from natural products as renin and angiotensin converting enzyme inhibitors. RSC Adv. 4 (2014) 19550-19568. [26] J. Xie, X. Chen, J. Wu, Y. Zhang, Y. Zhou, L. Zhang, Y. Tang, D. Wei, Antihypertensive effects, molecular docking study and isothermal titration calorimetry

assay of the angiotensin I-converting enzyme inhibitory peptides from Chlorella vulgaris. J. Agric. Food Chem. 66 (2018) 1359-1368. [27] H. Fujita, K. Yokoyama, M. Yoshikawa, Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food

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proteins. J. Food Sci. 65 (2000) 564-569. [28] O. Abdelhedi, R. Nasri, L. Mora, M. Jridi, F. Toldrá, M. Nasri, In silico analysis

SC R

and molecular docking study of angiotensin I-converting enzyme inhibitory peptides

from smooth-hound viscera protein hydrolysates fractionated by ultrafiltration. Food

U

Chem. 239 (2018), 453-463.

N

[29] X. Duan, F. Wu, M. Li, N. Yang, C. Wu, Y. Jin, J. Yang, Z. Jin, X. Xu, Naturally

A

occurring angiotensin I-converting enzyme inhibitory peptide from a fertilized egg

M

and its inhibitory mechanism. J. Agric. Food Chem. 62 (2014) 5500-5506.

ED

[30] M. Guo, X. Chen, Y. Wu, L. Zhang, W. Huang, Y. Yuan, M. Fang, J. Xie, D. Wei, Angiotensin I-converting enzyme inhibitory peptides from Sipuncula (Phascolosoma

PT

esculenta): Purification, identification, molecular docking and antihypertensive effects

CC E

on spontaneously hypertensive rats. Process Biochem. 63 (2017) 84-95. [31] Q. Wu, J. Du, J. Jia, C. Kuang, Production of ACE inhibitory peptides from sweet sorghum grain protein using alcalase: hydrolysis kinetic, purification and molecular

A

docking study. Food Chem. 218 (2016) 140-149. [32] J. R. Maple, M. Hwang, K. J. Jalkanen, T. P. Stockfisch, U. Dinur, M. Waldman, C. S. Ewig, A. T. Hagler, Derivation of class II force fields: V. Quantum force field for amides, peptides, and related compounds. J. Comp. Chem. 15 (1994) 162-182.

[33] T. J. Lin, H. Heinz, Accurate force field parameters and pH resolved surface models for hydroxyapatite to understand structure, mechanics, hydration, and biological interfaces. J. Phys. Chem. C. 120 (2016) 4975-4992. [34] P. Sadatmousavi, E. Kovalenko, P. Chen, Thermodynamic characterization of the

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interaction between a peptide-drug complex and serum proteins. Langmuir 30 (2014) 11122-11130.

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[35] M. C. Araujo, R. L. Melo, M. H. Cesari, M. A. Juliano, A. Luiz Juliano, A. K.

Carmona, Peptidase specificity characterization of C- and N-terminal catalytic sites of

U

angiotensin I-converting enzyme. Biochem. 39 (2000) 8519-8525.

N

[36] A. J. Turner, N. M. Hooper, The angiotensin-converting enzyme gene family:

A

genomics and pharmacology. Trends Pharmacol. Sci. 23 (2002) 177-183.

M

[37] M. S. Orloff, A. J. Turner, N. W. Bunnett, Catabolism of substance P and

ED

neurotensin in the rat stomach wall is susceptible to inhibitors of angiotensin converting enzyme. Regul. Peptides 14 (1986) 21-31.

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[38] S. Cosconati, S. Forli, A. L. Perryman, R. Harris, D. S. Goodsell, A. J. Olson,

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Virtual screening with AutoDock: theory and practice. Expert Opin. Drug Dis. 5 (2010) 597-607.

[39] J. S. Kim, M. Ree, T. J. Shin, O. H. Han, S. J. Cho, Y. T. Hwang, J. Y. Bae, J. M.

A

Lee, R. Ryoo, H. Kim, X-ray absorption and NMR spectroscopic investigations of zinc glutarates prepared from various zinc sources and their catalytic activities in the copolymerization of carbon dioxide and propylene oxide. J. Catal. 218 (2003) 209219.

Fig. 1. ITC profile of the three peptides and Lisinopril binding to ACE. (a) Lisinopril; (b) RYDF ; (c) YASGR ; (d) GNGSGYVSR. Fig. 2. ACE inhibitory activity variation of three peptides during the incubation with ACE within 24 h.

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Fig. 3. HPLC spectra of the three peptides before and 4 h after the incubation with ACE.

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Fig. 4. Identification of the hydrolysis product of GNGSGYVSR by UPLC & Q-TOF MS.

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Fig. 5. Time course of hydrolysis of GNGSGYVSR by ACE determined by HPLC.

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Fig. 6. Lineweaver-Burk plots of ACE inhibition by SR. 1/V and 1/S represents the

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reciprocal of reaction velocity and substrate concentration, respectively.

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Fig. 7. The specific interactions of GNGSGYVSR and its ACE hydrolysis products

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with ACE by molecular docking. (a) The 2D diagram of interactions between SR and ACE; (b) The 2D diagram of interactions between GNGSGYV and ACE; (c) The 2D

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diagram of interactions between GNGSGYVSR and ACE. H-bonds with main chains

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of amino acids of ACE are represented by a green dashed arrow directed towards the electron donor. H-bonds with side-chains of amino acids ACE are represented by a blue dashed arrow directed towards the electron donor.

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Fig. 8. Superimpose of the best binding conformation of peptides docked into the active site of ACE. (a) Local overview of the three peptides within the binding pockets. SR (yellow), GNGSGYV (purple) and GNGSGYVSR (brown) are shown in

stick model. S1 and S1’ pockets are displayed in blue and white surface; (b) Detailed interactions between peptides and ACE.

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Fig. 9. CD spectra of ACE and ACE-peptide complexes.

Table 1 Thermodynamic parameters and dissociation constant of three peptides binding to ACE determined by ITC -TS

H

G

Kd

(cal/mol·K)

(kcal/mol)

(kcal/mol)

(kcal/mol)

(nmol)

GNGSGYVSR

59.7

-16.6

3.12

-13.5

0.12

YASGR

56.8

-15.8

4.46

-11.4

4.50

RYDF

11.9

-3.55

-2.45

-6.00

16000

lisinopril

9.17

-2.73

-10.4

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-13.1

N A M ED PT CC E A

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S

Peptides

0.089

Table 2

Peptides

ΔG (KJ/mol)

IC50 (μM)

GNGSGYV

-9.78

ND

SR

-10.8

790 ± 64.2

GNGSGYVSR

-13.3

25 ± 3.7

SR (+ GNGSGYV)

-11.9

GNGSGYV (+ SR)

-7.89

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170 ± 7.2

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The ACE inhibitory IC50 values of GNGSGYVSR and its hydrolysis products

Table 3 Kinetics parameters of SR binding with ACE in different concentration 0 mg/mL

0.5 mg/mL

1.0 mg/mL

Vmax (mg/mL•min)

4.56 ± 0.28

4.56 ± 0.28

4.56 ± 0.28

Km (mM)

6.49 ± 0.15

1.51 ± 0.21

0.66 ± 0.09

Ki (mM)

2736 ± 56.2

2736 ± 56.2

2736 ± 56.2

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SR

Table 4 Secondary structure information of ACE and ACE-peptide complexes. ACE

ACE+GNGSGYVSR

ACE+SR

ACE+GNGSGYV

ACE+SR+GNGSGYV

α-helices

11.3%

17.6%

12.3%

15.3%

9.8%

β-sheets

22.7%

21.7%

21.1%

20.7%

16.7%

Parallel

10.5%

10.6%

12.6%

11.2%

Beta-Turn

9.2%

10.9%

10.2%

9.9%

Random Coils

46.3%

39.1%

43.8%

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Secondary structure

42.9%

14.9% 10.3% 48.2%

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Fig. 1.

(a)

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(d)

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Fig. 2.

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(b)

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Fig. 3.

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M (c)

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Fig. 4.

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A

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Fig. 5.

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Fig. 6.

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(b)

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Fig. 7.

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M (c)

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(b)

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Fig. 8.

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Fig. 9.