Abstract 237: Structural Analysis of Ang Peptide Specificity in Binding to ACE, ACE2, Neprilysin, and PRCP

Abstract

A combination of known biochemistry and physiology data on angiotensin converting enzyme (ACE), ACE2, neprilysin and prolylcarboxypeptidase (PRCP) with structural biochemistry and bioinformatics reveals several key amino acids contributing to the orientation of Ang peptides in the active site of the various enzymes. A total of four structures are known for the ACE N-terminus, twelve for the ACE C-terminus, eleven for ACE2, seven for neprilysin, and one for PRCP. Of all the known structures, none include angiotensin peptides bound into an active site. Autodock experiments reveal possible binding conformations of Ang I, Ang II, Ang-(1-9), Ang-(1-7), or Ang III into the active site of each enzyme. These autodock experiments were validated using combinations of disease associated natural variants, known mutagenesis data, conservation of amino acids in multiple species and inhibitor based structural data, all revealing amino acids of high functionality that interact with Ang peptides. Aromatic amino acids (Phe, Tyr, and Trp) tend to dictate the cleavage site of the Ang peptides through interactions with amino acid 4 (Tyr), 6 (His) or 8 (Phe) of angiotensin. The ACE C-terminus and N-terminus have three conserved aromatic amino acids 10 Å or less from the catalytic Zn, ACE2 has two 11 Å or less from the catalytic Zn, neprilysin has a Phe 8.4Å from the catalytic Zn, and PRCP has five aromatic amino acids 12Å or less from the active site. A high density of serine and threonine amino acids is found in the active sites of some enzymes when compared with total serines and threonines (ACE N-term=39%, ACE C-term=46%, ACE2=32%, neprilysin=15%, PRCP=10%), suggesting a strong likelihood of a hydrogen bonding network with angiotensin peptides in the active site, aiding in proper orientation for cleavage. Positions of serines and threonines (along with Lys, Arg, Val, Tyr, Phe, and Met) differ in the two active sites of ACE and are in contact with docked Ang I. This study shows enzyme differences and similarities in amino acids contacting the Ang peptides. Further, our results suggest that it may be possible to target multiple enzymes with the same (or a single) treatment in hypertensive patients, minimizing additive side effects of multidrug treatments.