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Inhibition mechanism and model of an angiotensin I-converting enzyme (ACE)-inhibitory hexapeptide from yeast (Saccharomyces cerevisiae).

Ni H, Li L, Liu G, Hu SQ - PLoS ONE (2012)

Bottom Line: The hexapeptide was found to inhibit ACE in a non-competitive manner, as supported by the structural model.The displacement of the zinc ion from the active site resulted in the inhibition of ACE activity.This study provides a new inhibitory mechanism of ACE by a peptide which broads our knowledge for drug designing against enzyme targets.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, Guangdong, China.

ABSTRACT
Angiotensin I-converting enzyme (ACE) has an important function in blood pressure regulation. ACE-inhibitory peptides can lower blood pressure by inhibiting ACE activity. Based on the sequence of an ACE-inhibitory hexapeptide (TPTQQS) purified from yeast, enzyme kinetics experiments, isothermal titration calorimetry (ITC), and a docking simulation were performed. The hexapeptide was found to inhibit ACE in a non-competitive manner, as supported by the structural model. The hexapeptide bound to ACE via interactions of the N-terminal Thr1, Thr3, and Gln4 residues with the residues on the lid structure of ACE, and the C-terminal Ser6 attracted the zinc ion, which is vital for ACE catalysis. The displacement of the zinc ion from the active site resulted in the inhibition of ACE activity. The structural model based on the docking simulation was supported by experiments in which the peptide was modified. This study provides a new inhibitory mechanism of ACE by a peptide which broads our knowledge for drug designing against enzyme targets.

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The ITC titration curve.A. Binding of HHL to ACE at pH 8.3. B. Binding of HHL to ACE and TPTQQS at pH 8.3.
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pone-0037077-g002: The ITC titration curve.A. Binding of HHL to ACE at pH 8.3. B. Binding of HHL to ACE and TPTQQS at pH 8.3.

Mentions: Non-competitive inhibition is a system in which the inhibitor and the substrate may both be bound to the enzyme at any given time. When both the substrate and the inhibitor are bound, the enzyme-substrate-inhibitor complex cannot form a product but can only be converted back into the enzyme-substrate complex or the enzyme-inhibitor complex [23]. Thus, ITC experiments were carried out to determine whether TPTQQS is a competitive inhibitor or a non-competitive inhibitor of ACE. As shown in Fig. 2, the titration curves showed a remarkable difference between the addition of HHL to solutions of ACE in the presence or absence of the inhibitor (TPTQQS). In the absence of the inhibitor TPTQQS, the enzymatic reaction showed a typical titration curve (Fig. 2A). When HHL was titrated into the ITC cell, it could bind to ACE and be hydrolyzed to HA by ACE. Initially, the titrated HHL bound to ACE completely, resulting in a higher binding enthalpy (ΔH) and producing a high titration peak. With the increase in the amount of HA, the binding of HHL to ACE was decreased by product inhibition [24], and the reaction tended toward equilibrium. Thus, the peaks corresponding to the raw heat rate gradually decreased in size. Full saturation was achieved at the end of the titration, and background heat of dilution was observed. When TPTQQS bound to ACE in the ITC cell (Fig. 2B), the ITC curve was remarkably different from that for the control experiment in which the peaks for the raw heat rate did not decrease remarkably. In addition, the affinity of HHL for ACE was the same as that in the control experiment, indicating that TPTQQS might act at a different site from the HHL binding site and thus HHL could bind to ACE continuously during the titration experiment [25]. On the basis of these results, we speculated that the binding of TPTQQS to ACE does not prevent the binding of HHL to ACE. Thus, when both HHL and TPTQQS were bound to ACE, the enzyme-substrate-inhibitor complex could not yield HA and could only be converted back into the enzyme-inhibitor complex, allowing another binding event between HHL and ACE. These ITC results are remarkably different from those for a system involving competitive inhibition [26], further verifying that the inhibition mechanism of ACE by TPTQQS is non-competitive.


Inhibition mechanism and model of an angiotensin I-converting enzyme (ACE)-inhibitory hexapeptide from yeast (Saccharomyces cerevisiae).

Ni H, Li L, Liu G, Hu SQ - PLoS ONE (2012)

The ITC titration curve.A. Binding of HHL to ACE at pH 8.3. B. Binding of HHL to ACE and TPTQQS at pH 8.3.
© Copyright Policy
Related In: Results  -  Collection

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pone-0037077-g002: The ITC titration curve.A. Binding of HHL to ACE at pH 8.3. B. Binding of HHL to ACE and TPTQQS at pH 8.3.
Mentions: Non-competitive inhibition is a system in which the inhibitor and the substrate may both be bound to the enzyme at any given time. When both the substrate and the inhibitor are bound, the enzyme-substrate-inhibitor complex cannot form a product but can only be converted back into the enzyme-substrate complex or the enzyme-inhibitor complex [23]. Thus, ITC experiments were carried out to determine whether TPTQQS is a competitive inhibitor or a non-competitive inhibitor of ACE. As shown in Fig. 2, the titration curves showed a remarkable difference between the addition of HHL to solutions of ACE in the presence or absence of the inhibitor (TPTQQS). In the absence of the inhibitor TPTQQS, the enzymatic reaction showed a typical titration curve (Fig. 2A). When HHL was titrated into the ITC cell, it could bind to ACE and be hydrolyzed to HA by ACE. Initially, the titrated HHL bound to ACE completely, resulting in a higher binding enthalpy (ΔH) and producing a high titration peak. With the increase in the amount of HA, the binding of HHL to ACE was decreased by product inhibition [24], and the reaction tended toward equilibrium. Thus, the peaks corresponding to the raw heat rate gradually decreased in size. Full saturation was achieved at the end of the titration, and background heat of dilution was observed. When TPTQQS bound to ACE in the ITC cell (Fig. 2B), the ITC curve was remarkably different from that for the control experiment in which the peaks for the raw heat rate did not decrease remarkably. In addition, the affinity of HHL for ACE was the same as that in the control experiment, indicating that TPTQQS might act at a different site from the HHL binding site and thus HHL could bind to ACE continuously during the titration experiment [25]. On the basis of these results, we speculated that the binding of TPTQQS to ACE does not prevent the binding of HHL to ACE. Thus, when both HHL and TPTQQS were bound to ACE, the enzyme-substrate-inhibitor complex could not yield HA and could only be converted back into the enzyme-inhibitor complex, allowing another binding event between HHL and ACE. These ITC results are remarkably different from those for a system involving competitive inhibition [26], further verifying that the inhibition mechanism of ACE by TPTQQS is non-competitive.

Bottom Line: The hexapeptide was found to inhibit ACE in a non-competitive manner, as supported by the structural model.The displacement of the zinc ion from the active site resulted in the inhibition of ACE activity.This study provides a new inhibitory mechanism of ACE by a peptide which broads our knowledge for drug designing against enzyme targets.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, Guangdong, China.

ABSTRACT
Angiotensin I-converting enzyme (ACE) has an important function in blood pressure regulation. ACE-inhibitory peptides can lower blood pressure by inhibiting ACE activity. Based on the sequence of an ACE-inhibitory hexapeptide (TPTQQS) purified from yeast, enzyme kinetics experiments, isothermal titration calorimetry (ITC), and a docking simulation were performed. The hexapeptide was found to inhibit ACE in a non-competitive manner, as supported by the structural model. The hexapeptide bound to ACE via interactions of the N-terminal Thr1, Thr3, and Gln4 residues with the residues on the lid structure of ACE, and the C-terminal Ser6 attracted the zinc ion, which is vital for ACE catalysis. The displacement of the zinc ion from the active site resulted in the inhibition of ACE activity. The structural model based on the docking simulation was supported by experiments in which the peptide was modified. This study provides a new inhibitory mechanism of ACE by a peptide which broads our knowledge for drug designing against enzyme targets.

Show MeSH