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

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.