The Angiotensin I-Converting Enzyme Inhibitory Peptides Of Peanut (Arachis Hypogaea L.) Storage Proteins: Properties and Structure based Inhibitor Design

Jimsheena, V. K. (2010) The Angiotensin I-Converting Enzyme Inhibitory Peptides Of Peanut (Arachis Hypogaea L.) Storage Proteins: Properties and Structure based Inhibitor Design. PhD thesis, University of Mysore.

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Abstract

Angiotensin I-converting enzyme (ACE; peptidyl dipeptide hydrolase, EC 3.4.15.1) plays a pivotal role in blood pressure regulation. Current attention focuses on mechanisms of controlling blood pressure through the inhibition of ACE. Bioactive antihypertensive peptides of food origin are increasingly gaining importance as alternates to synthetic drugs in hypertension therapy. Most of these antihypertensive peptides have been characterized by ACE inhibitor assay based on the hydrolysis of the synthetic peptide hippuryl-histidyl-leucine (HHL) to hippuric acid (HA) and histidyl-leucine (HL). The extent of HA released is directly proportional to the ACE activity. The reported assays for ACE are time consuming, cumbersome and not suitable for routine screening of large number of potential ACE inhibitor peptides present in food protein hydrolysates and numerous plant extracts. A diagnostic colorimetric assay for urine HA was transformed in to an ACE and a highthroughput ACE inhibitor assay. The released HA when complexed with pyridine and benzene sulfonyl chloride forms a yellow color, which is measured at 410 nm. This method is simple, sensitive, and rapid, requiring no solvent extraction and can therefore be used for high-throughput screening of ACE inhibitors. The limit of detection and limit of quantitation are at the nanomolar level. The application of this method for the detection of several ACE inhibitor peptides guaranteed by the enzymatic hydrolysis of arachin, the major storage protein of Arachis hypogaea has been demonstrated. In addition intra-molecularly fluorescence quenched peptide ‘Mca-LVPRGSKEFHL-Dnp’ was designed for use as a universal substrate for protease assay and has applications in fingerprinting aberrant proteases in tissues. The applicability and versatility of the substrate was demonstrated with a diverse array of physiological and several other commercially available proteases. The use of this universal substrate alleviates the need for multiple substrates to suit the cleavage specificity of individual proteases. The ACE inhibitory property of a Protease-P digest of arachin, the major storage globulin of peanut (Arachis hypogaea) has been demonstrated. The ACE inhibitory activity of a tripeptide (IEY) isolated from these digests has been characterized. Five synthetic structural analogs of this peptide (IEW, IKY, IKW, IEP and IKP) were assembled and their ACE inhibitory activity evaluated. Among these, the tripeptide IKP was a potent competitive inhibitor with an IC50 of 7 ± 1 × 10−6 M similar to that of the potent whey peptides IPP and VPP. The inhibition data of these peptide analogs have been rationalized through docking simulations using the tACE– lisinopril complex at 2Å resolution (PDB: 1086). The best docking poses were located at the tACE catalytic site resembling the mode of inhibition exerted by lisinopril, the synthetic drug. The degree of inhibition by the peptides correlated with the coordination distance between the catalytic Zn (II) and the carbonyl oxygen of the peptide bond between the amino-terminal and middle residue. These studies illustrate that these peptides, like lisinopril, behave as transition state analog inhibitors and are useful in therapeutic intervention for blood pressure management. Physiological proteases pepsin, trypsin, chymotrypsin and pancreatin were used to release antihypertensive peptides from arachin. The degree of proteolysis and in vitro ACE inhibition was maximum with pepsin. The ACE inhibitor index of human gastric juice catalyzed digestion was similar to pepsin demonstrating that such peptides can be produced in vivo following ingestion of arachin. Three antihypertensive peptides purified from the simulated gastric fluid digests of arachin were synthesized. Among them, the pentapeptide, NAQRP was the most potent with an IC50 of 32 ± 2 μM. A molecular docking study of NAQRP-tACE complexation, although stabilized by similar H-bond and hydrophobic interactions reveals subtle differences when compared to the previous tripeptide interactions. Computer simulation of the C-terminal tripeptide, QRP-tACE interaction suggested increased ACE binding affinity reckoned by its registry at the catalytic cavity by coordinating Zn (II) in semblance to the tripeptide, IKP. The prediction was validated by synthesizing the tripeptides QRP. The inhibitory potential was two-fold higher than NAQRP as predicted. These results suggest that in addition to the favorable residues, Zn (II) coordination distance and the length of the peptides are important for ACE inhibitor potency. These results promote the use of arachin/peanut proteins as functional ingredients in auxiliary therapeutic foods toward blood pressure management. A database, ACEpepDB, was created using PHP scripting language (version 5.2.6), MySQL relational database management system (version 5.0.5) and Apache web server (version 2.2) for web interfacing. It hosts detailed information on 853 peptides derived from 128 food sources, 14 assay methods used to monitor antihypertensive activity and 10 purification methods. Bibliographic data related to each of these interfaces are also listed. ACEpepDB can be searched using keywords such as peptide, mass, food source, assay method, purification method and reference details. The database ACEpepDB is a useful tool to retrieve preliminary information on all aspects of food derived antihypertensive peptide.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Angiotensin I-converting enzyme, blood pressure, Bioactive antihypertensive peptides, storage protein, Arachis hypogaea, arachin
Subjects:	500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 25 Peptide Chemistry 600 Technology > 08 Food technology > 16 Nutritive value > 03 Proteins
Divisions:	Protein Chemistry and Technology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	01 Mar 2011 05:13
Last Modified:	01 Mar 2011 05:13
URI:	http://ir.cftri.res.in/id/eprint/9942

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