O. A. Kost

Evidence for the negative cooperativity of the two active sites within bovine somatic angiotensin-converting enzyme

The somatic isoform of angiotensin‐converting enzyme (ACE) consists of two homologous domains (N‐ and C‐domains), each bearing a catalytic site. We have used the two‐domain ACE form and its individual domains to compare characteristics of different domains and to probe mutual functioning of the two active sites within a bovine ACE molecule. The substrate Cbz‐Phe‐His‐Leu (N‐carbobenzoxy‐L‐phenylalanyl‐L‐histidyl‐L‐leucine; from the panel of seven) was hydrolyzed faster by the N‐domain, the substrates FA‐Phe‐Gly‐Gly (N‐(3‐[2‐furyl]acryloyl)‐L‐phenylalanyl‐glycyl‐glycine) and Hip‐His‐Leu (N‐benzoyl‐glycyl‐L‐histidyl‐L‐leucine) were hydrolyzed by both domains with equal rates, while other substrates were preferentially hydrolyzed by the C‐domain. The inhibitor captopril ((2S)‐1‐(3‐mercapto‐2‐methylpropionyl)‐L‐proline) bound to the N‐domain more effectively than to the C‐domain, whereas lisinopril ((S)‐N α‐(1‐carboxy‐3‐phenylpropyl)‐L‐lysyl‐L‐proline) bound to equal extent with all ACE forms. However, active site titration with lisinopril assayed by hydrolysis of FA‐Phe‐Gly‐Gly revealed that 1 mol of inhibitor/mol of enzyme abolished the activity of either two‐domain or single‐domain ACE forms, indicating that a single active site functions in bovine somatic ACE. Neither of the k cat values obtained for somatic enzyme was the sum of k cat values for individual domains, but in every case the value of the catalytic constant of the hydrolysis of the substrate by the two‐domain ACE represented the mean quantity of the values of the corresponding catalytic constants obtained for single‐domain forms. The results indicate that the two active sites within bovine somatic ACE exhibit strong negative cooperativity.

Temperature-induced selective death of the C-domain within angiotensin-converting enzyme molecule

Somatic angiotensin‐converting enzyme (ACE) consists of two homologous domains, each domain bearing a catalytic site. Differential scanning calorimetry of the enzyme revealed two distinct thermal transitions with melting points at 55.3 and 70.5°C. which corresponded to denaturation of C‐ and N‐domains, respectively. Different heat stability of the domains underlies the methods of acquiring either single active N‐domain or active N‐domain with inactive C‐domain within parent somatic ACE. Selective denaturation of C‐domain supports the hypothesis of independent folding of the two domains within the ACE molecule. Modeling of ACE secondary structure revealed the difference in predicted structures of the two domains, which, in turn, allowed suggestion of the region 29–133 in amino acid sequence of the N‐part of the molecule as responsible for thermostability of the N‐domain.

New feature of angiotensin-converting enzyme: Carbohydrate-recognizing domain

Self carbohydrate‐mediated dimerization of glycoprotein angiotensin‐converting enzyme (ACE) was demonstrated. The dimerization was studied in the reverse micelle experimental system as a model of biomembrane situation. Asialo‐ACE or agalacto‐ACE was able to form a dimer, whereas deglycosylated ACE and sequentially desialylated and degalactosylated ACE failed to dimerize. ACE–ACE interaction was competitively inhibited by Neu5Ac‐ or Gal‐terminated saccharides. The results have allowed us to propose the existence of carbohydrate‐recognizing domain (CRD) on ACE molecule. The structural requirements of this CRD were estimated based on the ability of saccharides to inhibit ACE dimerization. The most effective monosaccharides with equal inhibition potencies were shown to be galactose (as GalβOMe) and N‐acetylneuraminic acid (as Neu5AcαOMe). Among oligosaccharides, the most effective ones were found to be 3′SiaLac and, especially, the whole pool of ACE oligosaccharide chains and biantennae complex oligosaccharide chains of other glycoproteins. Bovine and human ACEs were shown to be similar in terms of recognition of carbohydrates. Copyright

Simultaneous Determination of ACE Activity with 2 Substrates Provides Information on the Status of Somatic ACE and Allows Detection of Inhibitors in Human Blood

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of 2 homologous domains, each bearing a Zn-dependent active site. The ratio of the rates of hydrolysis of 2 synthetic substrates, Z-Phe-His-Leu (ZPHL) and Hip-His-Leu (HHL), is characteristic for each type of ACE: somatic 2-domain 1, N-domain 4.5, and C-domain 0.7 (Williams et al, 1996). We hypothesized that inactivation or selective inhibition of the C-domain within the somatic ACE should increase the ratio from 1 toward higher values, whereas inactivation or inhibition of the N-domain should decrease the ratio to lower values. Temperatures in the 40-60°C range cause preferential inactivation of the C-domain in somatic ACE and an increase in the ZPHL/HHL ratio. Determination of the ZPHL/HHL ratio in freshly 100-fold concentrated urine ruled out the existence of the N-domain fragment in human urine, which appears only as a result of long storage. Inhibition of ACE by common inhibitors also increases the ZPHL/HHL ratio for 2-domain ACE, thus providing a sensitive method for the detection of ACE inhibitors in biological fluids. Therefore, simultaneous measurements of ACE activity with 2 substrates (ZPHL and HHL) and calculation of their ratio allow us to monitor the status of the ACE molecule and detect ACE inhibitors (endogenous and exogenous) in human blood and other biological fluids. This method should find wide application in monitoring clinical trials with ACE inhibitors and in the development of the approach for personalized medicine by these effective drugs.

GLYCOSYLATION OF BOVINE PULMONARY ANGIOTENSIN-CONVERTING ENZYME MODULATES ITS CATALYTIC PROPERTIES

To study the role of the oligosaccharide moiety in the catalytic properties of angiotensin‐converting enzyme (ACE), we obtained asialo‐ and partially deglycosylated ACE by enzymatic treatment of two‐domain somatic enzyme from bovine lung. Treated enzymes demonstrated appreciable, but different changes of catalytic properties in the reaction of the hydrolysis of N‐substituted tripeptides, C‐terminal analogs of angiotensin I and bradykinin among them, compared to those for native enzyme. Deglycosylation also altered the catalytic properties of a single N domain of bovine ACE. So, various patterns of glycosylation modulate substrate specificity of somatic ACE and may be the reason for functional heterogeneity of the enzyme.

Enzyme activation and inactivation induced by low doses of irradiation

Activation phenomenon has been observed with two sets of enzymes under the conditions of low dosage irradiation. Activation was registered for angiotensin-converting enzyme under in vitro γ-irradiation (0.662 MeV, pulse duration approx 10s) at dose levels of 1–3 Gy and under X-ray irradiation (approx 9 keV, pulseduration approx 10−9s) at dose levels of 2×10−5 Gy. An activation effect has also occurred for native and recombinant horseradish peroxidase and tobacco peroxidase under γ-irradiation. The phenomenon observed is rationalized in terms of a kinetic model suggesting the existence of at least one activated enzyme conformation induced by radiolysis. The activity oscillations registered in dense plasma focus experiments were rationalized using the same model with the corresponding kinetic equation converted into the form describing the decaying oscillations caused by exciting force. The model analysis is presented.

Lysozyme and bilirubin bind to ACE and regulate its conformation and shedding

Angiotensin I-converting enzyme (ACE) hydrolyzes numerous peptides and is a critical participant in blood pressure regulation and vascular remodeling. Elevated tissue ACE levels are associated with increased risk for cardiovascular and respiratory disorders. Blood ACE concentrations are determined by proteolytic cleavage of ACE from the endothelial cell surface, a process that remains incompletely understood. In this study, we identified a novel ACE gene mutation (Arg532Trp substitution in the N domain of somatic ACE) that increases blood ACE activity 7-fold and interrogated the mechanism by which this mutation significantly increases blood ACE levels. We hypothesized that this ACE mutation disrupts the binding site for blood components which may stabilize ACE conformation and diminish ACE shedding. We identified the ACE-binding protein in the blood as lysozyme and also a Low Molecular Weight (LMW) ACE effector, bilirubin, which act in concert to regulate ACE conformation and thereby influence ACE shedding. These results provide mechanistic insight into the elevated blood level of ACE observed in patients on ACE inhibitor therapy and elevated blood lysozyme and ACE levels in sarcoidosis patients.

Tissue Specificity of Human Angiotensin I-Converting Enzyme.

Background Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. Methods/Principal Findings We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. Conclusions Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs.

Conformational Changes of Blood ACE in Chronic Uremia

Background The pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes. Hypothesis Toxic substances in the blood of patients with uremia due to End Stage Renal Disease (ESRD) can induce local conformational changes in the ACE protein globule and alter the efficacy of ACE inhibitors. Methodology/Principal Findings The recognition of ACE by 16 mAbs to the epitopes on the N and C domains of ACE was estimated using an immune-capture enzymatic plate precipitation assay. The precipitation pattern of blood ACE by a set of mAbs was substantially influenced by the presence of ACE inhibitors with the most dramatic local conformational change noted in the N-domain region recognized by mAb 1G12. The “short” ACE inhibitor enalaprilat (tripeptide analog) and “long” inhibitor teprotide (nonapeptide) produced strikingly different mAb 1G12 binding with enalaprilat strongly increasing mAb 1G12 binding and teprotide decreasing binding. Reduction in S-S bonds via glutathione and dithiothreitol treatment increased 1G12 binding to blood ACE in a manner comparable to enalaprilat. Some patients with uremia due to ESRD exhibited significantly increased mAb 1G12 binding to blood ACE and increased ACE activity towards angiotensin I accompanied by reduced ACE inhibition by inhibitory mAbs and ACE inhibitors. Conclusions/Significance The estimation of relative mAb 1G12 binding to blood ACE detects a subpopulation of ESRD patients with conformationally changed ACE, which activity is less suppressible by ACE inhibitors. This parameter may potentially serve as a biomarker for those patients who may need higher concentrations of ACE inhibitors upon anti-hypertensive therapy.

Role of two chloride-binding sites in functioning of testicular angiotensin-converting enzyme.

Modeling the structure of the C-domain of bovine angiotensin-converting enzyme revealed two putative chloride-binding sites. The kinetic parameters, Km and kcat, of hydrolysis of the substrate Cbz-Phe-His-Leu catalyzed by the testicular (C-domain) enzyme were determined over a wide range of chloride concentrations. Chloride anions were found to be enzyme activators at relatively low concentrations, but they inhibit enzymatic activity at high concentrations. A general scheme for the effect of chloride anions on activity of the C-domain of bovine angiotensin-converting enzyme accounting for binding the “activating” and “ inhibiting” anions is suggested.