Activation of angiotensin converting enzyme by monovalent anions

Abstract

The angiotensin converting enzyme catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions in the order C1- greater than Br- greater than F- greater than NO3- greater than CH3COO-. In the alkaline pH region, increasing anion concentrations decrease the KM but do not change the kcat. This behavior is characteristic of an ordered bireactant mechanism in which the anion binds to the enzyme prior to the substrate. At acidic pH values, however, the anion activation is a result of both a decrease in KM and an increase in kcat, implying a bireactant mechanism in which anion and substrate bind randomly. For both the ordered and the bireactant mechanisms the anion serves as an essential activator. The effect of chloride on enzyme activity was studied over the pH range 5-10 under kcat/KM conditions and demonstrates that the apparent chloride binding constant increases from 3.3 mM at pH 6.0 to 190 mM at pH 9.0. The kcat vs. pH profile exhibits two pK values of 5.6 and 9.6, while the variation of KM with pH is characterized by a pK of 8.9 and a 2-fold increase between pH 6.5 and 7.5. The chloride activation of the hydrolysis of furanacryloyl-Phe-Gly-Gly is compared with that of the physiological substrates angiotensin I and bradykinin.

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

“…Since its discovery, ACE was thought to be a metaldependent peptidase because enzymatic activity was virtually totally inhibited by EDTA (Skeggs et al, 1956a;Bünning and Riordan, 1983). The purified enzyme was shown to contain zinc by atomic absorption spectroscopy, and it was estimated (incorrectly) A Modern Understanding of ACE 5 that each molecule of ACE contained only one metal atom (Das and Soffer, 1975;Bünning and Riordan, 1985).…”

Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85

“…In this regard, the Cl--stimulated cellobiosidase again resembles the arylsulfatase, which showed little change in its pH activity curve when the chloride content was varied (42). Previous studies on anionactivated enzymes appear to point to the presence of two distinct types of activation (7). Bovine lysosomal cathepsin C (18) and Escherichia coli Mg,Ca-ATPase (1) belong to a group of anion-activated enzymes in which anions only affect the K,,,.…”

“…Only the activation of a-amylase seems to be as effective, and the maximal activity of this enzyme was found at anion concentrations below 5 mM (30). Many other chloride-activated enzymes show their highest activities at relatively high anion concentrations (1,18,38,42 Results of studies on several anion-stimulated enzymes have indicated that activation is accompanied by a shift of the pH activity profile toward the alkaline pH region, presumably because the combination of the anion with a basic group adjacent to a basic iotlizable group at the active site shifts the dissociation constant of the ionizable group toward the alkaline side (7,11,41). This does not seem to apply to the activation of the cellobiosidase.…”

An improved fluorometric assay of rat serum and plasma converting enzyme.

“…21 n Figure 1 shows that there was a moderate concentration-dependent activation by borate that was demonstrable even in the presence of a near optimal NaCl concentration (0.9 M) at pH 8.3 (see Table 3). Ionic strength has been suggested as an explanation for activation by sodium sulfate 18 ; however, Bunning and Riordan 17 and Shapiro and colleagues' 9 have shown that the activation achieved by increasing the concentration of some anions appears to be a specific effect and is not due only to ionic strength. In contrast to borate, increasing concentrations of phosphate in the presence of 0.3 M NaCl increased and then decreased both human and rat serum ACE activity (Figure 1).…”