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Angiotensin Converting Enzyme (ACE) Site‐directed Mutations Designed to Study the Enzyme Interaction with its Specific Inhibitors
Author(s) -
Aragão Danielle,
Pereira Larissa,
Affonso Regina,
Reis Rosana,
Casarini Dulce
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.1041.2
Subject(s) - enzyme , mutant , biochemistry , amino acid , renin–angiotensin system , chemistry , mutation , angiotensin converting enzyme , biology , gene , endocrinology , blood pressure
ACE is considered a key enzyme of the renin angiotensin system (RAS), which is involved in blood pressure regulation and electrolyte and fluid homeostasis. ACE is responsible for generating the most important vasoconstrictor peptide of the RAS, angiotensin II. The use of ACE inhibitors and antagonists of angiotensin II receptors are strategies for the treatment of hypertension. Considering its physiological importance, ACE has being the target of many studies which seek for better understanding of its structure and its interaction with specific inhibitors. The aim of this study is the development of site‐directed mutations in order to investigate whether modifications in the target amino acid residues may compromise the enzyme/inhibitor interaction through the purification and characterization of ACE mutants. The target amino acid residues were chosen based on the structural studies of the enzyme and are strong candidates for interaction with ACE inhibitors. Oligonucleotides for the change of the target amino acids were synthesized to the complementary strands containing the desired mutation of the coding region and were expressed in mammal cells using pcDNA plasmid containing human ACE cDNA. PCR reactions were performed in order to promote the site‐directed mutations. The DNA purified from transformed bacteria was sequenced to analyze the success of the mutation. The development of these site‐directed mutations and further biochemical characterization of purified ACE mutants could highlight the importance of these amino acid residues for the activity and/or function of the enzyme, possibly contributing to further identification of new ACE inhibitors. Supported by CAPES and FAPESP.