Abstract FR591: Enzymatic activity evaluation of site-directed mutations in the N-domain of Angiotensin-Converting Enzyme (ACE) and interaction with specific substrates

The Angiotensin-Converting Enzyme (ACE) features a cap region in its N-domain, composed of α-helices, which is critical for substrate and inhibitor interaction. This study investigated the role of conserved amino acids in this region regarding enzyme stability and functionality. Specific mutations (Ser11Val, Gln30Val, Arg53Ile, Ser61Ile, and Gln62Ala) were introduced, and their effects were analyzed under different temperature and pH conditions. The ACE cDNA, including the proposed mutations, was cloned into the pcDNA 3.1(+) vector and expressed in CHO cells. Wild-type and mutant enzymes were purified by gel filtration chromatography and evaluated for enzymatic activity using the substrates Hippuryl-His-Leu and ZPhe-His-Leu. Results indicated successful expression of the Ser11Val and Gln30Val mutants, which, despite exhibiting similar activity to the wild-type ACE, showed significant changes in substrate interaction under varying pH and temperature conditions. In conclusion, the cap region plays a crucial role in substrate-enzyme interaction, influencing ACE stability and activity, with potential clinical implications for the efficacy of ACE inhibitor therapies, particularly in patients harboring conformational-altering mutations.