Studies on the Interaction between Angiotensin-Converting Enzyme

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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]

1

ACS Paragon Plus Environment


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ABSTRACT

2

Angiotensin converting enzyme (ACE) inhibitory peptides derived from food

3

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

4

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

7

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

9

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

10

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

12

understanding of the inhibitory mechanism of ACE inhibitory peptides.

13 14

KEYWORDS: Angiotensin converting enzyme inhibitory peptides; static quenching

15

interaction; molecular docking; isothermal titration calorimetry; circular dichroism

16

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.

27

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

28

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

29

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

30

antihypertensive drugs.

3, 4

It plays an

31

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

32

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

33

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

34

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

36

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

37

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

38

preventing hypertension.

39

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

40

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

41

limited.

42

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

43

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

3



13

45

peptides have been reported. Fu et al.

46

peptides via fluorescence and simulated their interactions usingmolecular docking.

47

Xie et al.

48

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

49

These studies have provided information about the binding of ACE inhibitory

50

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

51

systematic thermodynamic, spectroscopic and computational studies on the interaction

52

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

53

reputation earned by spectroscopic, calorimetric and molecular docking techniques in

54

studying the protein-ligand interactions,

55

investigating the interaction between ACE inhibitory peptide and ACE.

56

14

analyzed the inhibition of ACE by patatin

studied the interaction mechanisms of TTW and VHW with ACE

In our previous work,

18

15-17

it is worth exploring their role in

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

57

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

58

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

60

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

61

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

62

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

63

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

64

could be beneficial for understanding the pharmacological and structural changes

65

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

69

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.

72

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

73

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

78

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

79

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

80

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

81

according to Michaelis-Menten kinetics.

82

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

83

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

84

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

85

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

86

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

87

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

88

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



89

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

90

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

91

interaction.

92

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

93

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

94

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

95

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

96

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

98

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

99

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

100

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

101

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

102

alone.

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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

113

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

114

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

115

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

116

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

117

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

118

CD spectroscopic measurements. CD spectral data were obtained from a

119

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

120

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

121

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

122

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

124

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

125

using SELCON 3 method in Dicroprot 2000 software.

126

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

127

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

128

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

129

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

132

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

134

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

135

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

136

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

137

GNGSGYVSR

138

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

23

22

Some

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

139

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

140

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

141

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

142

analysis.

143

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

149

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

150

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

151

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

152

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,

154

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

156

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

157

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

158

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

159

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

160

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.

163

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

164

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

165

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

166

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

169

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

170

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

171

parameters accompanying the binding of GMKCAF to ACE are summarized in

172

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).

9



174

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

177

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

178

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

179

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

180

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

181

hydrophobic force.

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

via “nonspecific binding”.

191

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

193

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

194

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

195

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

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

211

results are provided in Fig. 4.

212

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

213

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

214

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

215

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

216

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

217

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



218

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

219

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

220

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

221

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

222

contribution to the stability of peptide bound with ACE.

223

Recently, numerous ACE inhibitory peptides have been identified from protein

224

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

225

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

226

inhibitory mechanisms of several ACE inhibitory peptides (competitive and

227

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

228

of 1:1.14,

229

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

230

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

231

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

34, 35

This binding model may explain the competitive inhibition behavior.

232

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

233

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

234

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

235

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

236

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

237

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

238

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

239

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

241

temperature indicating the exposure of buried aromatic amino acids residues.

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249

binding interaction changed the hydrophobicity around the amide bonds and reflected

250

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

251

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

252

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

253

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

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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



262

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

263

on the ACE structure. 37-39

264

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

265

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

266

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

267

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

268

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

269

F0/F=1+Kq∙τ0∙[Q]=1+KSV∙[Q]

(3)

270

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

271

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

272

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

273

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

274

[Q] is the concentration of the quencher.

275

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

276

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

277

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

278

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

279

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

281

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

282

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

283

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

285

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

286

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

287

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

288

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

289

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

290

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

292

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

297

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

298

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

299

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

300

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



306

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

307

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

308

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

310

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.

312

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.

314

16


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Funding

22


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Page 23 of 36

442


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

443

(51372043),

Guangxi

Natural

Science

Foundation

444

2017GXNSFBA198215, 2017GXNSFAA198289)

23


(2017GXNSFDA198052,


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


Page 24 of 36

Page 25 of 36


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

25



Page 26 of 36

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

26


Page 27 of 36


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

27


R 0.9989 0.9933 0.9135


Page 28 of 36

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

28


Page 29 of 36


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 )

29



Fig. 2

a

b

30


Page 30 of 36

Page 31 of 36


Fig. 3

a

b

c

31



Fig. 4

a

b

c

32


Page 32 of 36

Page 33 of 36


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


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


280

300

320


Page 34 of 36

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


200 F

320

440

300 250

150

0 300

420

nm

Wavelength(nm)

-2

[Q]

0

2

4

6

8 [Q]

34


10

12

14

16

18

500

Page 35 of 36


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)

35



Table of Contents Graphic (TOC)

36


Page 36 of 36

Studies on the Interaction between Angiotensin-Converting Enzyme

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