Substrate specificity and kinetic characteristics of angiotensin converting enzyme

Abstract

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.

Porcine pulmonary angiotensin I-converting enzyme—Biochemical characterization and spatial arrangement of the N- and C-domains by three-dimensional electron microscopic reconstruction

“…The catalytic efficiency (kcat/Km) of sACE was calculated to be 67.6 x 10 6 M -1· min -1 as shown in Table 2. This value is in good agreement with data obtained by Bünning et al (1983). Table 3 presents the Ki values for captopril and BPP9a, as determined by the graphical method of Dixon et al (1979).…”

Determinants of angiotensin II generation during converting enzyme inhibition.

“…The structure of the most widely used substrate, Hip-Gly-Gly, differs from that of Ang I, and its K M (5 mM) is much less favorable than that determined for Ang I (70 JIM). 3 The second substrate used in this study, Z-Phe-His-Leu, represents the C-terminal sequence of Ang I. Its N-terminal is protected against nonspecific hydrolysis.…”

Matrix Metalloproteinase Inhibition During the Development of Congestive Heart Failure

“…30 -32 This MMP inhibitor was also examined with respect to activity against angiotensin-converting enzyme (prepared from rabbit lung), 33 neutral endopeptidase (24.11, membrane fraction from Burkitt lymphoma cell line), 34 endothelin-converting enzyme (membrane fraction from CHO cells transfected with human endothelin-converting enzyme 1), 35 and tumor necrosis factor-␣ convertase (tumor necrosis factor-␣ release from stimulated leukocytes; PanLabs Inc). 36 Concentrations of up to 100 mol/L of PD166793 did not exhibit inhibitory activity against these proteolytic systems (Table 1).…”