Mutations in two specific residues of testicular angiotensin-converting enzyme change its catalytic properties.
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.
Sen
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I
Department of Cardiovascular Biology, Cleveland Clinic Foundation, Ohio 44195.
Kasturi
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Abdul Jabbar
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Sen
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GC
D000595:Amino Acid Sequence; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015870:Gene Expression; D005822:Genetic Vectors; D005971:Glutamates; D018698:Glutamic Acid; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D017706:Lisinopril / Q000378:metabolism; D008239:Lysine; D008297:Male; D016296:Mutagenesis; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine; D017354:Point Mutation; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*; D014162:Transfection; D014443:Tyrosine; D014616:Vaccinia virus
D000806:Angiotensin-Converting Enzyme Inhibitors; D005971:Glutamates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D014443:Tyrosine; D010649:Phenylalanine; D017706:Lisinopril; D007703:Peptidyl-Dipeptidase A; D008239:Lysine