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 aldehyde and ketone substrate analogues

Three classes of carbonyl-containing substrate analogues and partial substrate analogues have been tested for their ability to inhibit angiotensin converting enzyme. (4-Oxobutanoyl)-L-proline is proposed to occupy the S1' and S2' subsites on the enzyme, thus locating its aldehyde carbonyl group at the position of the active site zinc atom. This aldehyde is 70% hydrated in aqueous solution and could mimic a tetrahedral intermediate occurring during enzyme-catalyzed substrate hydrolysis, but its Ki is only 760 microM. Carbobenzoxy-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalaninal is proposed to occupy the S1 through S4 subsites on the other side of the zinc atom. Its weak Ki of 60 microM is nearly equipotent to its parent peptide terminating in phenylalanine. However, ketoace, (5RS)-(5-benzamido-4-oxo-6-phenylhexanoyl)-L-proline [Almquist, R.G., Chao, W.R., Ellis, M.E., & Johnson, H.L. (1980) J. Med. Chem. 23, 1392-1398], one of the third class of inhibitors proposed to occupy subsites S1 through S2' on both sides of the zinc atom, has a Ki of 0.0006 microM under our assay conditions, orders of magnitude more potent than its parent peptide. The carbonyl carbon of ketoace is less than 3% hydrated in aqueous solution as determined by carbon-13 nuclear magnetic resonance spectroscopy. If the hydrate is the species bound to converting enzyme, its Ki must be less than 18 pM. Ketoace is a slow-binding inhibitor of converting enzyme, but its overall Ki is dependent on its concentration and therefore prevents calculation of kinetic constants for slow binding.(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.     

The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism

Zinc is essential to the catalytic activity of angiotensin converting enzyme. The enzyme contains one g-atom of zinc per mole of protein. Chelating agents abolish activity by removing the metal ion to yield the inactive, metal-free apoenzyme. Zinc does not stabilize protein structure since the native and apoenzymes are equally susceptible to heat denaturation. Addition of either Zn2+, Co2+, or Mn2+ to the apoenzyme generates an active metalloenzyme; Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ fail to restore activity. The activities of the metalloenzymes follow the order Zn greater than Co greater than Mn. The protein binds Zn2+ more firmly than it does Co2+ or Mn2+. Hydrolysis of the chromophoric substrate, furanacryloyl-Phe-Gly-Gly, by the active metalloenzymes is subject to chloride activation; the activation constant is not metal dependent. Metal replacement mainly affects Kcat with very little change in Km, indicating that the role of zinc is to catalyze peptide hydrolysis.     

Angiotensin converting enzyme: substrate inhibition

Phosphate, borate, and Tris inhibit angiotensin converting enzyme (ACE), but HEPES buffer is inert. Measurements of substrate inhibition were made in HEPES buffer at pH 7.0 and 25 degrees C and 37 degrees C. Substrate inhibition was marked and goes to completion. A new equation for substrate inhibitions enables one, under favorable circumstances, to determine whether there is cooperativity in the binding of substrate to the inhibitory and active sites. Cooperativity does occur with ACE using Hipp-His-Leu as substrate. The kinetic parameters were measured (Km = 0.21 mM, K* = 0.65 mM at 37 degrees C). The enzyme concentration (1.94 X 10(-8) M) was determined by titration with lisinopril so that kcat (5 X 10(3) at 37 degrees C) could be determined. Using this value and the molecular weight the specific activity of ACE was calculated for different common buffers. The specific activity in HEPES calculated from Vmax was 33.7 units/mg at 37 degrees C.     

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.     

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.     

Species relationships for plasma angiotensin converting enzyme activity using a furanacryloyl tripeptide substrate

Angiotensin converting enzyme (ACE; EC 3.4.15.1) activities were compared in plasma samples obtained from three species using a furanacryloyl tripeptide substrate. The enzyme activity observed in Wistar rat plasma was higher than the activities observed in the other two species. Using this substrate, human and canine plasma enzyme activities were similar-unlike published data where hippuryl-histidyl-leucine was used as substrate.     

Triiodothyronine increases serum angiotensin converting enzyme

To determine whether elevated thyroid hormone is responsible for increased serum angiotensin converting enzyme in hyperthyroidism, 5 to 40 micrograms of 3,5,3'-triiodo-L-thyronine was administered orally and subcutaneously to female Swiss-Webster mice. Serum angiotensin converting enzyme was significantly increased in all animals given triiodothyronine compared to controls. Lung and kidney enzymes were moderately reduced in specific activity but unchanged in total activity due to increase in size of these organs. The results indicate that in hyperthyroidism, elevated thyroid hormone per se rather than the disease of the thyroid is responsible for elevated serum angiotensin converting enzyme.     

Isolation and characterization of an angiotensin converting enzyme substrate from vitellogenic ovaries of Neobellieria bullata

Vitellogenic ovaries of the gray fleshfly Neobellieria bullata contain a variety of unidentified substances that interact, either as a substrate or as an inhibitor, with angiotensin converting enzyme (ACE). We here report the isolation and characterization of the first ACE interactive compound hereof. This 1312.7 Da peptide with the sequence NKLKPSQWISL, is substrate to both insect and human ACE. It is a novel peptide that shows high sequence similarity to a sequence at the N-terminal part of dipteran yolk polypeptides (YPs). We propose to call it N. bullata ovary-derived ACE interactive factor or Neb-ODAIF. Both insect and human ACE hydrolyze Neb-ODAIF by sequentially cleaving off two C-terminal dipeptides. K(m) values of Neb-ODAIF and Neb-ODAIF(1-9) (NKLKPSQWI) for human somatic ACE (sACE) are 17 and 81 microM, respectively. Additionally, Neb-ODAIF(1-7) (NKLKPSQ) also interacts with sACE (K(m/i)=90 microM). These affinity-constants are in range with those of the physiological ACE substrates and suggest the importance of Neb-ODAIF and its cleavage products in the elucidation of the physiological role of insect ACE. Alternatively, they can serve as lead compounds in the development of new drugs against ACE-related diseases in humans.     

The angiotensin I converting enzyme.

The angiotensin I converting enzyme (kininase II; peptidyl dipeptidase; EC3.4.15.1) has a dual function: it converts angiotensin I to angiotensin II and it inactivates bradykinin. Lung, kidney, guinea pig plasma and testicles are among the richest sources of the enzyme. Vascular endothelial cells and bursh borders of renal proximal tubular cells contain high concentrations of the enzyme. The availability of synthetic peptide inhibitors was a great help in establishing the function of converting enzyme in normal and pathological conditions.     

New potent inhibitors of angiotensin converting enzyme

Using an earlier model of the favoured orientation of binding functions of angiotensin converting enzyme (ACE) inhibitors, it has been possible to postulate a new, 7,6-bicyclic system, based on hexahydropyridazine, which might be expected to have high potency. Some members of this system which have been synthesised have been shown to be very active ACE inhibitors, in vitro and in vivo.     

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.