Catalytic properties of the two active sites of angiotensin I-converting enzyme on the cell surface.
Angiotensin I converting enzyme is a zinc metallopeptidase that contains two very similar domains, each with an active site. Enzymatic studies of these active sites have always been performed on solubilized enzyme, although angiotensin I converting enzyme is a transmembrane ectopeptidase. The availability of transfected CHO cells expressing wild-type recombinant enzyme and mutants in which one of the two active sites has been inactivated by site-directed mutagenesis allowed the properties of each active site on the cell surface and the effect of anchorage and membrane environment to be studied. Both active centers are catalytically active in the cell membrane-anchored enzyme and convert angiotensin I to angiotensin II. Comparison of the kinetic parameters for the transfected cells with those for the purified enzymes reveals differences in Kcat but suggests that no major conformational changes of these active sites occur upon anchorage of the enzyme to the cell membrane. The chloride activation profiles show that the two domains in the cell-bound enzyme also undergo the same anion-induced conformational changes as in the solubilized enzyme.
Jaspard
E
E
Institut National de la Santé et de la Recherche Médicale U367, Paris, France.
Alhenc-Gelas
F
F
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D002712:Chlorides / Q000494:pharmacology; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D002712:Chlorides; D011994:Recombinant Proteins; D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A