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 labe l was studied by the fluorescence polarization technique. The dissociation constants Kd of the enzyme-inhibitor complexes in 50 mM Hepes-buffer, pH 7. 5, containing 150 mM NaCl and 1 muM ZnCl2 at 37 degreesC were (2.3 +/- 0.4) (.)10(-8), (2.1 +/- 0.3)(.)10(-8), and (2.1 +/- 0.2)(.)10(-8) M for two-dom ain somatic ACE, single-domain testicular ACE, and for the N-domain of the enzyme, respectively. The interaction of the enzyme with the inhibitor stro ngly depended on the presence of chloride in the medium, and the apparent d issociation 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 reacti on, 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 complexe s with the inhibitor were apparently first-order, and the dissociation rate constants were similar: (0.055 +/- 0.001) and (0.041 +/- 0.001) sect for t he N-domain and for testicular ACE, respectively.