Fluorescence polarization studies of different forms of angiotensin-converting enzyme.
The interaction of three forms of bovine angiotensin-converting enzyme (ACE) with the competitive peptide inhibitor lisinopril with a fluorescent label was studied using fluorescence polarization. The dissociation constants Kd of the enzyme-inhibitor complexes in 50 mM Hepes-buffer (pH 7.5) containing 150 mM NaCl and 1 microM ZnCl2 at 37 degrees C were (2.3 +/- 0.4).10(-8), (2.1 +/- 0.3).10(-8), and (2.1 +/- 0.2).10(-8) M for two-domain somatic ACE, single-domain testicular ACE, and for the N-domain of the enzyme, respectively. The interaction of the enzyme with the inhibitor strongly depended on the presence of chloride in the medium, and the apparent dissociation constant of the ACE-chloride complex was (1.3 +/- 0.2).10(-3) M for the somatic enzyme. The dissociation kinetics of the complex of the inhibitor with somatic ACE did not fit the kinetics of a first-order reaction, but it was approximated by a model of simultaneous dissociation of two complexes with the dissociation rate constants (0.13 +/- 0.01) sec(-1) and (0.026 +/- 0.001) sec(-1) that were present at approximately equal initial concentrations. The dissociation kinetics of the single-domain ACE complexes with the inhibitor were apparently first-order, and the dissociation rate constants were similar: (0.055 +/- 0.001) and (0.041 +/- 0.001) sec(-1) for the N-domain and for testicular ACE, respectively.
Voronov
S V
SV
School of Chemistry, Lomonosov Moscow State University, Moscow, 119899, Russia.
Binevski
P V
PV
Eremin
S A
SA
Kost
O A
OA
D000818:Animals; D002417:Cattle; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D017706:Lisinopril / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007703:Peptidyl-Dipeptidase A / Q000187:drug effects / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D017706:Lisinopril; D007703:Peptidyl-Dipeptidase A