Mutations in two specific residues of testicular angiotensin-converting enzyme change its catalytic properties

• Published in: The Journal of biological chemistry Vol. 268; no. 34; pp. 25748 - 25754
• Main Authors: Sen, I, Kasturi, S, Abdul Jabbar, M, Sen, G C
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 05.12.1993; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Angiotensin-Converting Enzyme Inhibitors - metabolism; Animals; Binding Sites; Binding, Competitive; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Vectors; Glutamates; Glutamic Acid; HeLa Cells; Humans; Hydrolases; Kinetics; Lisinopril - metabolism; Lysine; Male; Mutagenesis; Peptidyl-Dipeptidase A - biosynthesis; Peptidyl-Dipeptidase A - genetics; Peptidyl-Dipeptidase A - metabolism; Phenylalanine; Point Mutation; Rabbits; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Substrate Specificity; Testis - enzymology; Transfection; Tyrosine; Vaccinia virus; Enzyme; Active site; Site directed mutagenesis; Rabbit; Lagomorpha; Testicle; Vertebrata; Regulation(control); Mammalia; Enzymatic activity; Structure activity relation; Peptidyl-dipeptidase A; Hydrolases; Peptidyl-dipeptidases; Proteinases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)74453-6

Chemical modifications of 2 specific residues present in angiotensin-converting enzyme (ACE) result in its inactivation, thereby suggesting that these 2 residues may be important for its enzyme activity. We directly tested this hypothesis by substituting Tyr-236 with Phe and Lys-154 with Glu in rabbit testicular ACE (ACET) using site-directed mutagenesis of the corresponding cDNA. Wild type ACET, the two single mutants, and the double mutant were expressed in HeLa cells using the vaccinia virus-T7 polymerase expression system. The rate of synthesis, post-translational modifications, and cleavage secretion pattern of all four proteins were indistinguishable. The enzymatic properties of the two single mutants and the wild type enzyme were also very similar. In contrast, the double mutant had about a 20-fold lower specific activity although its Km was only 6-fold higher than that of the wild type protein. The double mutant also had a 100-fold higher Ki for lisinopril, a competitive inhibitor of ACET, and was 17-fold less sensitive to stimulation by NaCl, an activator of ACET. These results directly demonstrate that Tyr-236 and Lys-154 are indeed critical for the catalytic activity, lisinopril inhibition, and NaCl activation of ACET.