Activation of angiotensin I-converting enzyme by chloride ion as an allosteric activator

Abstract

Angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) exhibits a unique chloride ion requirement for its full catalytic activity, but its requirement of chloride ion was not absolute. The kinetic manifestation of chloride ion activation is characterized by a decrease in the apparent $K_m$ values for the substrates. The $V_\max$ value is not much affected. The optimum pH was shifted gadually to the alkaline region up to pH 8.3 depending on the concentration of chloride ion. When the enzyme was saturated with chloride ion, the $K_m$ values decreased by a factor of 50, while only an 18\% increase in $V_\max$ was observed. The $K_{Cl}$ value for the enzyme-chloride binding was estimated to be about 150 mM in all cases regardless of the peptide substrates employed. The effect of chloride ion on the molecular size of converting enzyme was studied by using several physicochemical techniques such as gel filtration, electrophoresis, and sucrose density gradient sedimentation analysis. As a result, it was found that there was no chance of the enzyme aggregation or dissociation by chloride ion, i.e. the mobilities of the enzyme in the exclusion chromatography, disc gel electrophoresis, and the sedimentation experiment were not much altered by the presence of chloride ion. The purified hog lung converting enzyme was homogeneous in S.D.S. polyacylamide gel electrophoresis and in 5-20\% w/v sucrose gradient sedimentation analysis. The enzyme contained no dissociable subunits under denatured conditions. The enzyme is found to be a single polypeptide chain, having a molecular weight approximately 150,000. In the presence of chloride ion, the enzyme causes its spectral changes. The ultraviolet spectrum of converting enzyme showed a blue shift of 2 nm accompanied by a 7\% increase in the intensity of the maximum absorption with 400 mM of NaCl. Chloride ion also affected the fluorescence emission spectra of the converting enzyme. In the absence of chloride io...