Catalysis of angiotensin I hydrolysis by human angiotensin-converting enzyme: effect of chloride and pH.
The catalysis of the hydrolysis of angiotensin I, an important natural substrate, by human angiotensin-converting enzyme (ACE) was examined in detail as a function of chloride and hydrogen ion concentration. Chloride was found to be a nonessential activator over the pH range 5.0-10.0, with the chloride dependence increasing with increasing pH: the velocity enhancement at optimal [Cl-] increased from 1.6- to 42-fold; the chloride optimum and Ka' increased from 20 to 520 mM and from 0.22 to 120 mM, respectively, and activity in the absence of chloride decreased from 60.9 to 2.4% (relative to maximal activation). Kinetic analyses at pH 6.0, 7.5, and 9.0 confirmed the nonessential activator mechanism. At all pH values tested chloride was found to be inhibitory (relative to maximal activation) at supraoptimal chloride levels. Depending on the [Cl-] range, both apparent uncompetitive and competitive modes were demonstrated. From pH 6.0 to 9.0 Kis varied between 110 and 1140 mM (apparent). In all cases Ki' much greater than Ka'. We suggest that at high [Cl-] chloride binds to low-affinity inhibitory sites on the free enzyme and on the ES and EP complexes. The pH-rate profile demonstrated a chloride-dependent alkaline shift, with the pH optimum increasing from 7.1 at zero chloride to 7.6 at 400 mM NaCl. At [S] much greater than Km a plot of log nu vs pH revealed pKs of 5.9 and 9.4 in the ES complex in the absence of chloride, while at maximally activating [Cl-] only one ionization at pK = 6.3 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Ehlers
M R
MR
Department of Medicine, University of Cape Town Medical School, Observatory, South Africa.
Kirsch
R E
RE
D000803:Angiotensin I / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D002712:Chlorides; D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A; D015032:Zinc