^{Biochemical and Kinetic Characterization of Angiotensin I Converting Enzymes (ACE) with Site‐Directed Amino Acid Changes: Interactions with Its Specific Inhibitors}

Abstract

ACE is responsible for generating the vasoconstrictor peptide angiotensin II of the Renin Angiotensin Aldosterone System, and blockage of this system with the use of ACE inhibitors and antagonists of angiotensin II receptors are strategies for treatment of hypertension. Studies demonstrated that the use of site‐directed mutagenesis and the construction of chimeric enzymes can assist in the identification of the amino acids responsible for the interaction of enzyme/substrate or enzyme‐inhibitor complexes. We investigated the role of amino acid residues targets located in the N‐domain of ACE, Ala361, Thr378 and Phe467, strong candidates for the interaction of the enzyme with its inhibitors. The enzyme mutants were expressed in CHO cells, purified and characterized by enzymatic activity using specific substrates, inhibition assays with captopril, lisinopril and enalapril. After plasmid expression of wild type ACE (WT) cDNA in mammalian cells, PCR was performed to insert mutations 1 to 9 (M1 to M9), followed by electrophoresis, bacterial transfection, amplification of plasmids and sequencing. Only mutations M4 (A361W), M7 (T378A) and M8 (F437A) showed satisfactory results. M4, M7, M8 and WT were purified by a Superdex 200 column coupled to the FPLC system. The purified enzymes were characterized biochemically through enzymatic assays using the specific substrates for ACE, Hippuryl‐His‐Leu (HHL) and Z‐Phe‐His‐Leu (z‐FHL). WT, M4 and M7 showed high specific activity values. Analyzing the catalytic efficiency (kcat/KM), we observed that mutant enzymes better hydrolyzed the substrate z‐FHL when compared to HHL. M7 showed higher specificity (0.39 mM) and catalytic velocity (1.02 s‐1) for the substrate z‐FHL. The one with the best specificity for the HHL substrate (1.35 mM) and catalytic velocity (0.54 s‐1) was M4, compared to the WT. All enzymes showed an optimum pH around 8.0 with both substrates. Captopril inhibited the enzymes most efficiently using HHL as substrate, while Lisinopril was the one that best inhibited the enzymes using z‐FHL. It suggests that there is a negative cooperation between the ACE catalytic sites by the substrate. For both inhibitors and substrates, the best inhibition occurred with M7, suggesting that this mutation may have altered the interaction of the enzyme active site. The results obtained in this work can lead to a better understanding of the importance of these amino acids residues for the activity/functionality of the enzyme, seeking the identification of new molecules that can inhibit ACE.