CATALYTIC PROPERTIES OF THE 2 ACTIVE-SITES OF ANGIOTENSIN-I-CONVERTING ENZYME ON THE CELL-SURFACE

Angiotensin I converting enzyme is a zinc metallopeptidase that contai ns two very similar domains, each with an active site. Enzymatic studi es of these active sites have always been performed on solubilized enz yme, although angiotensin I converting enzyme is a transmembrane ectop eptidase. The availability of transfected CHO cells expressing wild-ty pe recombinant enzyme and mutants in which one of the two active sites has been inactivated by site-directed mutagenesis allowed the propert ies of each active site on the cell surface and the effect of anchorag e and membrane environnement to be studied. Both active centers are ca talytically active in the cell membrane-anchored enzyme and convert an giotensin I to angiotensin II. Comparison of the kinetic parameters fo r the transfected cells with those for the purified enzymes reveals di fferences in Kcat but suggests that no major conformational changes of these active sites occur upon anchorage of the enzyme to the cell mem brane. The chloride activation profiles show that the two domains in t he cell-bound enzyme also undergo the same anion-induced conformationa l changes as in the solubilized enzyme. (C) 1995 Academic Press Inc.