Inhibition of angiotensin converting enzyme: mechanism and substrate dependence

The interaction of angiotensin converting enzyme with six metal-coordinating [(D-3-mercapto-2-methylpropanoyl)-L-Pro (captopril), N-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Pro (MK-422), N-(phenylphosphoryl)-L-Phe-L-Phe, N alpha-(3-mercaptopropanoyl)-L-Arg, N alpha-[1(S)-carboxy-3-phenylpropyl]-Ala-L-Lys, and N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly] and three dipeptide inhibitors (Gly-L-Trp, L-Phe-L-Arg, and L-Ala-L-Pro) was examined at pH 7.5 in the presence of 300 mM NaCl. Inhibition modes, apparent Ki [Ki(app)] values, and shapes of 1/v vs. [I] plots were found to vary with the substrate employed. All inhibitors except Phe-Arg were competitive with the substrate furanacryloyl (Fa)-Phe-Gly-Gly, while five of seven tested with Fa-Phe-Phe-Arg as substrate produced mixed patterns. Ki-(app) values for N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly, N-(phenylphosphoryl)-L-Phe-L-Phe, Gly-Trp, and MK-422 were 8.3-, 5.5-, 4.7-, and 2.6-fold lower, respectively, when Fa-Phe-Gly-Gly was substrate, compared with values measured with Fa-Phe-Phe-Arg. In contrast, Ki(app) values for Phe-Arg and (3-mercaptopropanoyl)-Arg were lower (2.8- and 2.2-fold, respectively) when Fa-Phe-Phe-Arg was the substrate. Plots of 1/v vs. [I] for most of the inhibitors were nonlinear, to an extent which was also substrate dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of angiotensin converting enzyme by phosphoramidates and polyphosphates

N alpha-Phosphoryl-L-alanyl-L-proline is a reversible competitive inhibitor of angiotensin converting enzyme with a Ki of 1.4 nM. Alkylation of one phosphate oxygen with methyl, ethyl, or benzyl does not change the Ki. The high activity of the O-alkylated inhibitors demonstrates that the two phosphate oxygen anions do not constitute a bidentate ligand of the active site zinc ion. Substitution of valyltryptophan, glycylglycine, or delta-aminovaleric acid for alanylproline in the phosphoramidate raises the Ki to 12 nM, 25 microM, and 178 microM, respectively. Methylation of the alanine nitrogen in phosphorylalanylproline raises the Ki to 29 microM. Polyphosphates inhibit converting enzyme with the following Ki's: phosphate, approximately 300 mM; pyrophosphate, 2 mM; tripolyphosphate, 18 microM; tetrapolyphosphate, 150 microM. The inhibition by tripolyphosphate appears to be competitive and is unaffected by the addition of excess zinc ion. Since the Ki of tripolyphosphate is nearly 10-fold lower than that of N-phosphoryl-delta-aminovaleric acid and is near that of N alpha-phosphorylglycylglycine, its terminal phosphates may bind the zinc site and the cationic site on the enzyme, thus spanning the S1' and S2' sites.     

Inhibition of angiotensin converting enzyme: dependence on chloride

In a previous report [Shapiro, R., Holmquist, B., & Riordan, J. F. (1983) Biochemistry 22, 3850], it was demonstrated that activation of angiotensin converting enzyme (ACE) by chloride is strongly dependent on substrate structure, and three substrate classes were identified on the basis of activation behavior. The present study examines the chloride dependence of the inhibition of ACE by nine inhibitors [(D-3-mercapto-2-methylpropanoyl)-L-Pro (captopril), N-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Pro (MK-422), L-Ala-L-Pro, N-(phenylphosphoryl)-L-Phe-L-Phe, Gly-L-Trp, N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly, L-Phe-L-Arg, N alpha-(3-mercaptopropanoyl)-L-Arg, and N alpha-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Lys] containing structural features characteristic of the three classes of substrates. Apparent Ki values for all inhibitors are markedly (70-250-fold) decreased by 300 mM chloride. However, the enhancement of inhibition is achieved at significantly lower chloride concentrations with those inhibitors having an ultimate arginine or lysine than with the remainder. This variability parallels that previously found for activation of substrate hydrolysis. The effect of chloride on the individual steps in the formation and dissociation of the steady-state enzyme-inhibitor complexes was determined with the slow-binding inhibitor MK-422. Pre-steady-state analysis indicates that binding of both MK-422 and captopril follows a (minimally) two-step mechanism: (formula; see text) in which rapid formation of an enzyme-inhibitor complex is followed by a slow isomerization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of angiotensin converting enzyme by the metalloendopeptidase 3.4.24.15 inhibitor c-phenylpropyl-alanyl-alanyl-phenylalanyl-p-aminobenzoate.

Inhibitors of metallopeptidases may represent new alternatives in the treatment of cardiovascular disease. Recent investigations have linked the hypotensive properties of the metalloendopeptidase 3.4.24.15 (MEP 24.15) inhibitor c-phenylpropyl-alanyl-alanyl-phenylalanyl-para-aminobenzoate (cFP-A-A-F-pAB) to the attenuation of bradykinin metabolism. However, since angiotensin converting enzyme (ACE) is widely recognized to contribute to the metabolic clearance of bradykinin, we characterized the specificity of cFP-A-A-F-pAB towards ACE. We also determined whether cFP-A-A-F-pAB inhibits the conversion of angiotensin I (Ang I) to Ang II by pulmonary ACE. The ACE activity toward the synthetic substrate hippuryl-histidine-leucine (Hip-His-Leu) was measured in vitro using both a purified lung preparation and pooled rat serum. The ACE activity was inhibited at increasing concentrations of the MEP 24.15 inhibitor. Kinetic analysis revealed that cFP-A-A-F-pAB competitively inhibited pulmonary ACE with a Ki of 0.19 microM. In rat serum, cFP-A-A-F-pAB also competitively inhibited ACE. The hydrolysis of Ang I into Ang II by pulmonary ACE was inhibited to a similar extent by both cFP-A-A-F-pAB and the ACE inhibitor MK 422. These findings are the first to show that the MEP 24.15 inhibitor cFP-A-A-F-pAB also inhibits ACE. We suggest that the reported hypotensive actions of cFP-A-A-F-pAB may be due to the reduction in both bradykinin metabolism and Ang II generation arising from the blockade of ACE.     

Inhibition of canine lung angiotensin converting enzyme by ACTH and structurally related peptides

Human ACTH and structurally related peptides, such as ACTH 7–38, ACTH 4–11, ACTH 1–10 and ACTH 18–39, noncompetitively inhibited the activity of angiotensin I converting enzyme (dipeptidyl carboxypeptidase; E.C. 3.4.15.1) in the preparation from canine lung. The Ki values were 1.5 μM and 0.54 μM for ACTH and ACTH 7–38, respectively, using[¹⁴C] -Hip-his-leu as the substrate. These results suggest that ACTH and ACTH 7–38 are potent inhibitors of angiotensin I converting enzyme without being substrate for the enzyme.     

Inhibition of angiotensin converting enzyme by L-alanyl-4 or 5-substituted-L-prolines and their N alpha-phosphoryl-derivatives

A series of L-alanyl-4 or 5-substituted L-prolines, such as L-alanyl-L-thiazolidine-4-carboxylic acid, L-alanyl-5-oxo-L-proline, L-alanyl-trans-4-hydroxy-L-proline and L-alanyl-cis-4-hydroxy-L-proline as well as their corresponding N alpha-phosphoryl derivatives, were synthesized and studied for inhibition against angiotensin converting enzyme. Furanacryloyl-phenylalanyl-glycyl-glycine was used as substrate in 50 mM Tris hydrochloride buffer at pH 7.5 containing 1 microM zinc acetate. N alpha-Phosphoryl-L-alanyl-L-thiazolidine-4-carboxylic acid and N alpha-phosphoryl-L-alanyl-trans-4-hydroxy-L-proline competitively inhibit angiotensin converting enzyme with Ki values of 68 microM and 89.3 microM, respectively. Smaller inhibition against angiotensin converting enzyme was obtained with the rest of compounds studied here.     

Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins

It was determined that flavan-3-ols and procyanidins have an inhibitory effect on angiotensin I converting enzyme (ACE) activity, and the effect was dependent on the number of epicatechin units forming the procyanidin. The inhibition by flavan-3-ols and procyanidins was competitive with the two substrates assayed: N-hippuryl-L-histidyl-L-leucine (HHL) and N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). Tetramer and hexamer fractions were the more potent inhibitors, showing Ki of 5.6 and 4.7 microM, respectively. As ACE is a membrane protein, the interaction of flavanols and procyanidins with the enzyme could be related to the number of hydroxyl groups on the procyanidins, which determine their capacity to be adsorbed on the membrane surface.     

Activation of angiotensin converting enzyme by monovalent anions

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.     

Inactivation of angiotensin converting enzyme by sodium nitroprusside

Angiotensin I converting enzyme is rapidly inactivated by sodium nitroprusside and that inactivation is suppressed in the presence of chloride ion and by the presence of L-alanyl-L-proline or glycyl-L-tryptophan, which are both competitive inhibitors of its catalytic activity. The inactivation by sodium nitroprusside appears to result from the modification of an unusually reactive lysine residue in or near the active site.     

Biochemical pharmacology of total retro-inverso analogues of bradykinin and angiotensin II: Molecular recognition by G-protein-coupled receptors and angiotensin converting enzyme

Summary Total retro-inverso (TRI) analogues of bradykinin (BK), the B_2a -selective kinin antagonistd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK, angiotensin II (AT II) and the AT II antagonist Saralasin ([Sar¹, Val⁵, Ala⁸]AT II) were prepared by conventional solid-phase synthesis. Molecular recognition of TRI peptidomimetics by G-protein-coupled receptors was studied by competitive radioligand displacement experiments. TRI analogues ofd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK specifically bound to the kidney medulla B_2a bradykinin receptor with affinities (K _d ) ranging from 64 μM to 4 μM. Conversely, TRI analogues of BK, AT II and Saralasin did not bind to either the B_2a bradykinin receptor or the rat AT_1a AT II receptor, respectively. These studies indicate that the TRI strategy is more compatible with the synthesis of antagonists than ‘agonists’. Three TRI peptidomimetics ofd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK were weak inhibitors of angiotensin converting enzyme. All other TRI peptidomimetics had no effect upon ACE activity. These data endorse the utility of the TRI strategy for the synthesis of protease-resistant antagonists of peptide hormones and neuropeptides.     

Perindopril and ramipril phosphonate analogues as a new class of angiotensin converting enzyme inhibitors

A series of phosphonate analogues related to perindopril and ramipril were prepared and tested to estimate their ability to inhibit angiotensin converting enzyme. These new synthesized compounds were active ACE inhibitors with a promising activity.     

Biochemical pharmacology of total retro-inverso analogues of bradykinin and angiotensin II: Molecular recognition by G-protein-coupled receptors and angiotensin converting enzyme

Total retro-inverso (TRI) analogues of bradykinin (BK), the B₂a-selective kinin antagonist d-Arg⁰[Hyp₃,d-Phe⁷,Leu⁸]BK, angiotensin II (AT II) and the AT II antagonist Saralasin ([Sar¹,Val⁵,Ala⁸]AT II) were prepared by conventional solid-phase synthesis. Molecular recognition of TRI peptidomimetics by G-protein-coupled receptors was studied by competitive radioligand displacement experiments. TRI analogues of d-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK specifically bound to the kidney medulla B₂a bradykinin receptor with affinities (K_d) ranging from 64 M to 4 M. Conversely, TRI analogues of BK, AT II and Saralasin did not bind to either the B_2a bradykinin receptor or the rat AT_1a AT II receptor, respectively. These studies indicate that the TRI strategy is more compatible with the synthesis of antagonists than agonists. Three TRI peptidomimetics of d-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK were weak inhibitors of angiotensin converting enzyme. All other TRI peptidomimetics had no effect upon ACE activity. These data endorse the utility of the TRI strategy for the synthesis of protease-resistant antagonists of peptide hormones and neuropeptides.     

Inhibition by angiotensin converting enzyme inhibitors of endothelin secretion from cultured human endothelial cells.

We conducted a study to determine whether angiotensin converting enzyme inhibitors (ACEIs) inhibit endothelin secretion from cultured human endothelial cells. Confluent umbilical vein endothelial cells were incubated in multi-well plates with culture medium containing either captopril (10(-6), 10(-5), 10(-4) M) or enalaprilat (10(-7), 10(-6), 10(-5) M) for 6 hours. Immunoreactive endothelin in the medium was measured by radioimmunoassay. Calf serum (CS) stimulated endothelin release in a concentration-dependent manner, and both ACEIs inhibited 5% CS-stimulated endothelin release in a concentration-dependent manner. To explore the mechanisms of ACEI-induced suppression of endothelin release, the effects of angiotensin II (10(-8), 10(-7), 10(-6) M), angiotensin converting enzyme (0.1, 1, 10 mU/ml), bradykinin (10(-8), 10(-7), 10(-6) M), and sodium nitroprusside (10(-6), 10(-5), 10(-4) M) on endothelin release were also examined. Although angiotensin II and angiotensin converting enzyme had no significant effect on endothelin release, concentration-dependent suppression occurred with bradykinin and sodium nitroprusside. These results indicate that ACEIs inhibit the stimulated release of endothelin from human endothelial cells, and provide indirect evidence that ACEI-induced ET suppression may be mediated via potentiation of autacoid formation from the cells.     

Inhibition of [3H]captopril binding by peptide analog angiotensin converting enzyme inhibitors

Ketomethylene containing peptide analogs, modeled after a snake venom pentapeptide, have been shown to be potent angiotensin converting enzyme inhibitors. Although the most potent compounds are up to five times more potent than captopril in inhibiting angiotensin converting enzyme activity, they are relatively weak inhibitors of [3H]captopril binding to membrane bound angiotensin converting enzyme. This indicates that inhibition of [3H]captopril binding and enzymatic activity is due to binding to distinct sites. These results suggest that the inhibitors bind to an additional site on the enzyme distinct from the captopril binding site.     

An angiotensin converting enzyme inhibitor is a tight-binding slow substrate of carboxypeptidase A

Carboxypeptidase A-catalyzed hydrolysis of peptides and depsipeptides is competitively inhibited by N-(1-carboxy-5-t-butyloxycarbonylaminopentyl)-L-phenylalanine (Boc-CA-Phe, Ki = 1.3 microM) and the angiotensin converting enzyme inhibitor, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine (Z-CA-Gly-Phe, Ki = 4.5 microM). The latter compound is actually a slow substrate of carboxypeptidase. Indirect observation of inhibitor binding by stopped-flow measurement of radiationless energy transfer between carboxypeptidase tryptophans and dansylated substrates reveals slow binding for both compounds. The visible absorption spectrum of the complex of cobalt(II)-substituted carboxypeptidase and Z-CA-Gly-Phe, which differs from the corresponding spectrum of the Boc-CA-Phe complex, is remarkable in its resemblance to the spectrum of the complex between Co(II)carboxypeptidase and a transient intermediate previously observed during hydrolysis of peptide substrates. The spectrum slowly changes to that of the free enzyme indicating hydrolysis. Chromatographic quantitation of substrate and products confirms that carboxypeptidase converts Z-CA-Gly-Phe to Z-CA-Gly and L-Phe with an apparent kcat of 0.02 s-1. Absorption spectroscopy indicates that the Z-CA-Gly-Phe-Co(II)carboxypeptidase spectrum is not that of bound products. Moreover, spectral titrations indicate that the products (both with spectral Ki values of about 3 mM), as well as D-Phe, compete for the same site on the enzyme.     

Purification of bovine angiotensin converting enzyme

A change has been made in the commonly used lisinopril affinity gel procedure for purifying angiotensin converting enzyme. The new method greatly decreases the time required and greatly increases the yield of pure enzyme. All of the enzyme in various bovine tissues was extracted with 0.5% triton X-100 and applied to the affinity column; 70% was trapped and all of the trapped enzyme was released as the apoenzyme by EDTA. The holoenzyme was recovered by dialysis against zinc containing buffer. The turnover numbers were precisely the same for enzyme from lung, atrium, kidney, striatum and blood. The tissue concentrations of ACE were very different but the final specific activities were the same.