Effect of inhibitors on the coordination geometries of cadmium at the metal sites in angiotensin-I-converting enzyme.
Perturbed angular correlation of gamma-rays (PAC) spectroscopy has been used to investigate the angiotensin-I-converting enzyme (ACE) of rabbit lung. By substituting the zinc ions in ACE with excited 111mCd2+ ions, analysis of PAC spectra gave directly the percentage of cadmium ions bound to ACE. The result of the analysis was a dissociation constant of about 1 microM for the cadmium-ACE complex, and a stoichiometry of two moles cadmium/mole enzyme. Cadmium binding is thus about two orders of magnitude weaker than zinc binding to ACE but two orders of magnitude stronger than cobalt binding. PAC spectra monitor the nuclear quadrupole interaction (NQI) for 111mCd. The NQI for ACE exhibits very low frequencies in the PAC spectra with a rather large spectral broadening. In the presence of the inhibitor ramiprilat, the frequencies increase but the spectral broadening is about the same as for ACE without inhibitor. When the inhibitor captopril is added, very high frequencies are obtained consistent with sulfur binding, but now with a narrower distribution of NQI's. A simple molecular orbital analysis of the obtained NQI's has been performed, using a coordination sphere of two His, one Glu residue and a solvent ligand, equivalent to the zinc ligands in thermolysin and carboxypeptidase. The calculated spectral parameters could be modelled with the measured parameters if the solvent ligand is H2O in free ACE, carboxylate from ramiprilat in the ACE-ramiprilat complex and a mercapto group in the ACE-captopril complex. The coordination geometry for cadmium carboxypeptidase obtained by X-ray diffraction gives a calculated set of NQI parameters consistent with the measured parameters for cadmium in the captopril-ACE complex using a mercapto group as the solvent ligand. However, for ACE and its complex with ramiprilat, a significant distortion of the cadmium geometry for carboxypeptidase A had to be adopted in order to calculate NQI's close to the experimental values.
Carvalho
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Department of Biochemistry and Biophysics, University of Göteborg, Sweden.
Göthe
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Bauer
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Danielsen
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Hemmingsen
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D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism*; D002216:Captopril / Q000494:pharmacology; D002268:Carboxypeptidases / Q000737:chemistry; D005720:Gamma Rays; D008168:Lung / Q000201:enzymology; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D017257:Ramipril / Q000031:analogs & derivatives / Q000494:pharmacology; D013057:Spectrum Analysis; D015032:Zinc / Q000378:metabolism
D000806:Angiotensin-Converting Enzyme Inhibitors; D002104:Cadmium; C052549:ramiprilat; D002216:Captopril; D002268:Carboxypeptidases; D007703:Peptidyl-Dipeptidase A; D015032:Zinc; D017257:Ramipril