Substrate specificity and kinetic characteristics of angiotensin converting enzyme

Furanacryloyl-Phe-Gly-Gly has been shown to be a convenient substrate for angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1). A detailed kinetic analysis of the hydrolysis of this substrate indicates normal Michaelis-Menten behavior with kcat = 19000 min-1 and KM = 3.0 x 10(-4) M determined at pH 7.5, 25 degrees C. The enzyme is inhibited by phosphate and activated by chloride; maximal activity is observed with 300 mM NaCl. In the absence of added zinc, activity is lost rapidly below pH 7.5 due to spontaneous dissociation of the metal, but in the presence of zinc, the enzyme remains fully active to about pH 6. The pH-rate profile indicates two groups on the enzyme with apparent pK values of 5.6 and 8.4. The substrate specificity of the enzyme has been examined in terms of the fundamental specificity quantity kcat/KM as well as the separate constants by using a series of furanacryloyl-tripeptides. The activity toward furanacryloyl-Phe-Gly-Gly has been compared with that toward the physiological substrates angiotensin I and bradykinin.