Functional Evaluation Of Angiotensin‐Converting Enzyme (ACE) Amino Acid Residues important for the interaction with its inhibitors

Abstract

Hypertension is a worldwide health problem considered to be one of the greatest public health challenges. Since hypertension is a major risk factor for cardiovascular and renal disease, many studies have focused on the Renin‐Angiotensin System (RAS), an endocrine system where renin cleaves angiotensinogen and releases Angiotensin I (AI) which is converted into Angiotensin II (AII) by the action of Angiotensin Converting Enzyme I (ACE). The ACE has a large physiological importance being a target of several therapeutic studies. This work aims to investigate the role of amino acids residues Asp140, Gln259, Ala332, Ser 333, Gln 355, Thr 358, Phe435 and Arg 500, in the interaction with the ACE inhibitor lisinopril as well as other ones.. An analysis of sequence homology to the ACEs of Xenopus laevis, mouse, rat and human, human isoform N‐domain (ND) and isoforms of 65 and 90 kDa of rat showed a high conservation of amino acid residues proposed to be mutated. Primers containing the mutation were synthesized. Following, we used a vector containing the cDNA with the human ACE gene (pACE) to perform PCR reactions; then the products obtained from PCR reactions were digested with the enzyme DpnI and transformed in E. coli DH5α. Positive colonies had the plasmid extracted, sequenced and analyzed concerning the desired mutation. The ACE mutants will be analyzed related to enzyme‐substrate interaction as well as enzyme activity.