Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme

Quenched fluorescence peptides were used to investigate the substrate specificity requirements for recombinant wild-type angiotensin I-converting enzyme (ACE) and two full-length mutants bearing a single functional active site (N- or C-domain). We assayed two series of bradykinin-related peptides flanked by o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp), namely, Abz-GFSPFXQ-EDDnp and Abz-GFSPFRX-EDDnp (X = natural amino acids), in which the fluorescence appeared when Abz/EDDnp are separated by substrate hydrolysis. Abz-GFSPFFQ-EDDnp was preferentially hydrolyzed by the C-domain while Abz-GFSPFQQ-EDDnp exhibits higher N-domain specificity. Internally quenched fluorescent analogues of N-acetyl-SDKP-OH were also synthesized and assayed. Abz-SDK(Dnp)P-OH, in which Abz and Dnp (2,4-dinitrophenyl) are the fluorescent donor-acceptor pair, was cleaved at the D-K(Dnp) bond with high specificity by the ACE N-domain (k(cat)/K(m) = 1.1 microM(-)(1) s(-)(1)) being practically resistant to hydrolysis by the C-domain. The importance of hydroxyl-containing amino acids at the P(2) position for N-domain specificity was shown by performing the kinetics of hydrolysis of Abz-TDK(Dnp)P-OH and Abz-YDK(Dnp)P-OH. The peptides Abz-YRK(Dnp)P-OH and Abz-FRK(Dnp)P-OH which were hydrolyzed by wild-type ACE with K(m) values of 5.1 and 4.0 microM and k(cat) values of 246 and 210 s(-)(1), respectively, have been shown to be excellent substrates for ACE. The differentiation of the catalytic specificity of the C- and N-domains of ACE seems to depend on very subtle variations on substrate-specific amino acids. The presence of a free C-terminal carboxyl group or an aromatic moiety at the same substrate position determines specific interactions with the ACE active site which is regulated by chloride and seems to distinguish the activities of both domains.     

Monoclonal Antibodies 1G12 and 6A12 to the N-domain of human angiotensin-converting enzyme: fine epitope mapping and antibody-based detection of ACE inhibitors in human blood

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N- and C-), each bearing a Zn-dependent active site. ACE inhibitors are among the most prescribed drugs in the treatment of hypertension and cardiac failure. Fine epitope mapping of two monoclonal antibodies (mAb), 1G12 and 6A12, against the N-domain of human ACE, was developed using the N-domain 3D-structure and 21 single and double N-domain mutants. The binding of both mAbs to their epitopes on the N-domain of ACE is significantly diminished by the presence of the C-domain in the two-domain somatic tissue ACE and further diminished by the presence of sialic acid residues on the surface of blood ACE. The binding of these mAbs to blood ACE, however, increased dramatically (5-10-fold) in the presence of ACE inhibitors or EDTA, whereas the effect of these compounds on the binding of the mAbs to somatic tissue ACE was less pronounced and even less for truncated N-domain. This implies that the binding of ACE inhibitors or removal of Zn2+ from ACE active centers causes conformational adjustments in the mutual arrangement of N- and C-domains in the two-domain ACE molecule. As a result, the regions of the epitopes for mAb 1G12 and 6A12 on the N-domain, shielded in somatic ACE by the C-domain globule and additionally shielded in blood ACE by sialic acid residues in the oligosaccharide chains localized on Asn289 and Asn416, become unmasked. Therefore, we demonstrated a possibility to employ these mAbs (1G12 or 6A12) for detection and quantification of the presence of ACE inhibitors in human blood. This method should find wide application in monitoring clinical trials with ACE inhibitors as well as in the development of the approach for personalized medicine by these effective drugs.     

Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4: tolerance of a CDR peptide to conformational changes of a target

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 degrees 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.     

Selective inhibition of the C-domain of angiotensin I converting enzyme by bradykinin potentiating peptides

Somatic angiotensin I converting enzyme (ACE) contains two functional active sites. Up to now, most of the studies aimed at characterizing the selectivity of inhibitors toward the two ACE active sites relied on the use of ACE mutants containing a single functional active site. By developing new fluorogenic synthetic substrates of ACE, we demonstrated that inhibitor selectivity can be assessed directly by using somatic ACE. This useful screening approach led us to discover that some bradykinin potentiating peptides turned out to be selective inhibitors of the C-domain of ACE. The peptide pGlu-Gly-Leu-Pro-Pro-Arg-Pro-Lys-Ile-Pro-Pro, with K(i)(app) values of 30 nM and 8 microM, respectively, for the C- and N-domain of ACE, is to our knowledge the most highly selective C-domain inhibitor of ACE so far reported. Inhibitors able to block selectively either the N- or C-domain of ACE will represent unique tools to probe the function of each domain in the regulation of blood pressure or other physiopathological events involving ACE activity.     

Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild-type angiotensin-converting enzyme

Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild-type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE.     

Structural determinants of RXPA380, a potent and highly selective inhibitor of the angiotensin-converting enzyme C-domain

RXPA380 (Cbz-PhePsi[PO(2)CH]Pro-Trp-OH) was reported recently as the first highly selective inhibitor of the C-domain of somatic angiotensin-converting enzyme (ACE), able to differentiate the two active sites of somatic ACE by a selectivity factor of more than 3 orders of magnitude. The contribution of each RXPA380 residue toward this remarkable selectivity was evaluated by studying several analogues of RXPA380. This analysis revealed that both pseudo-proline and tryptophan residues in the P(1)' and P(2)' positions of RXPA380 play a critical role in the selectivity of this inhibitor for the C-domain. This selectivity is not due to a preference of the C-domain for inhibitors bearing pseudo-proline and tryptophan residues, but rather reflects the poor accommodation of these inhibitor residues by the N-domain. A model of RXPA380 in complex with the ACE C-domain, based on the crystal structure of germinal ACE, highlights residues that may contribute to RXPA380 selectivity. From this model, striking differences between the N- and C-domains of ACE are observed for residues defining the S(2)' pocket. Of the twelve residues that surround the tryptophan side chain of RXPA380 in the C-domain, five are different in the N-domain. These differences in the S(2)' composition between the N- and C-domains are suggested to contribute to RXPA380 selectivity. The structural insights provided by this study should enhance understanding of the factors controlling the selectivity of the two domains of somatic ACE and allow the design of new selective ACE inhibitors.     

Structure and physiological importance of angiotensin converting enzyme domains

Somatic angiotensin converting enzyme (ACE) consists of two homologous catalytic domains (N- and C-domain), exhibiting different biochemical properties. The catalytically active ACE isoforms consisted of just one domain have been also detected in mammals. Substantial progress in ACE domain research was achieved during the last years, when their crystal structures were determined. The crystal structures of domains in complex with diverse potent ACE inhibitors provided new insights into structure-based differences of the domain active sites. Physiological functions of ACE are not limited by regulation of the cardiovascular system. Recent evidence suggests that the ACE domains may be also involved into control of different physiological functions. The C-terminal catalytic domain plays an important role in the regulation of blood pressure: it catalyzes angiotensin I cleavage in vivo. The N-domain contributes to the processing of other bioactive peptides for which it exhibits high affinity. The role of the N-domain is not ultimately associated with functioning of the rennin-angiotensin system and it contributes processing of other bioactive peptides for which it exhibits high affinity (goralatide, luliberin, enkephalin heptapeptide, beta-amyloid peptide). Domain-selective inhibitors selectively blocking either the N- or C-domain of ACE have been developed.     

Isolation and characterization of the N-domain of bovine angiotensin-converting enzyme

A method for preparation of a catalytically active fragment of bovine lung angiotensin-converting enzyme (ACE) has been developed. It includes limited proteolysis of the full-length somatic form of the enzyme by trypsin. The resulting fragment corresponds to the N-terminal domain of angiotensin-converting enzyme. The influence of chloride and sulfate anions on the enzymatic activity of this fragment has been investigated, and kinetic parameters for hydrolysis of synthetic tripeptide substrates catalyzed by the N-domain of ACE have been determined. Comparison of these parameters with those obtained for full-length somatic bovine ACE suggests that in the bovine somatic ACE molecule active centers located in various domains may function interdependently.     

Hydrolysis by somatic angiotensin-I converting enzyme of basic dipeptides from a cholecystokinin/gastrin and a LH-RH peptide extended at the C-terminus with gly-Arg/Lys-arg, but not from diarginyl insulin.

Endoproteolytic cleavage of protein prohormones often generates intermediates extended at the C-terminus by Arg-Arg or Lys-Arg, the removal of which by a carboxypeptidase (CPE) is normally an important step in the maturation of many peptide hormones. Recent studies in mice that lack CP activity indicate the existence of alternative tissue or plasma enzymes capable of removing C-terminal basic residues from prohormone intermediates. Using inhibitors of angiotensin I-converting enzyme (ACE) and CP, we show that both these enzymes in mouse serum can remove the basic amino acids from the C-terminus of CCK5-GRR and LH-RH-GKR, but only CP is responsible for converting diarginyl insulin to insulin. ACE activity removes C-terminal dipeptides to generate the Gly-extended peptides, whereas CP hydrolysis gives rise to CCK5-GR and LH-RH-GK, both of which are susceptible to the dipeptidyl carboxypeptidase activity of ACE. Somatic ACE has two similar protein domains (the N-domain and the C-domain), each with an active site that can display different substrate specificities. CCK5-GRR is a high-affinity substrate for both the N-domain and C-domain active sites of human sACE (Km of 9.4 microm and 9.0 microm, respectively) with the N-domain showing greater efficiency (kcat : Km ratio of 2.6 in favour of the N-domain). We conclude that somatic forms of ACE should be considered as alternatives to CPs for the removal of basic residues from some Arg/Lys-extended peptides.     

Angiotensin I-converting enzyme transition state stabilization by HIS1089: evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His(1089) as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His(1089) to Ala or Leu. At pH 7.5, with Ang I as substrate, k(cat)/K(m) values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (k(cat)) with minor effects on ground state substrate binding (K(m)). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His(1089) --> Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His(1089) side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His(1089) on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.     

Functional conservation of the active sites of human and Drosophila angiotensin I-converting enzyme

Human somatic angiotensin I-converting enzyme (sACE) has two active sites present in two homologous protein domains, resulting from a tandem gene duplication. It has been proposed that the N- and C-terminal active sites can have specific in vivo roles. In Drosophila melanogaster, Ance and Acercode for two ACE-like single-domain proteins, also predicted to have distinct physiological roles. We have investigated the relationship of Ance and Acer to the N- and C-domains of human sACE by genomic sequence analysis and by using domain-selective inhibitors, including RXP 407, a selective inhibitor of the human N-domain. These phosphinic peptides were potent inhibitors of Acer, but not of Ance. We conclude that the active sites of the N-domain and of Acer share structural features that permit the binding of the unusual RXP407 inhibitor and the hydrolysis of a broader range of peptide structures. In comparison, Ance, like the human C-domain of ACE, displays greater inhibitor selectivity. From the analysis of the published sequence of the Adh region of Drosophila chromosome 2, which carries Ance, Acer, and four additional ACE-like genes, we also suggest that this functional conservation is reflected in an ancestral gene structure identifiable in both protostome and deuterostome lineages and that the duplication seen in vertebrate genomes predates the divergence of these lineages. The conservation of ACE enzymes with distinct active sites in the evolution of both vertebrate and invertebrate species provides further evidence that these two kinds of active sites have different physiological functions.     

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides for defining substrate specificity of the angiotensin I-converting enzyme and development of selective C-domain substrates

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides were used for the analyses of the S(3) to S(1)' subsites of the somatic angiotensin I-converting enzyme (ACE). Substrate specificity of ACE catalytic domains (C- and N-domains) was assessed in an effort to design selective substrates for the C-domain. Initially, we defined the S(1) specificity by preparing a library with the general structure Abz-GXXZXK(Dnp)-OH [Abz = o-aminobenzoic acid, K(Dnp) = N(epsilon)-2,4-dinitrophenyllysine, and X is a random residue], where Z was successively occupied with one of the 19 natural amino acids with the exception of Cys. The peptides containing Arg and Leu in the P(1) position had higher C-domain selectivity. In the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH, Arg was fixed at P(1) so we could define the C-domain selectivity of the S(1)', S(2), and S(3) subsites. On the basis of the results from these libraries, we synthesized peptides Abz-GVIRFK(Dnp)-OH and Abz-GVILFK(Dnp)-OH which contain the most favorable residues for C-domain selectivity. Systematic reduction of the length of these two peptides resulted in Abz-LFK(Dnp)-OH, which demonstrated the highest selectivity for the recombinant ACE C-domain (k(cat)/K(m) = 36.7 microM(-1) s(-1)) versus the N-domain (k(cat)/K(m) = 0.51 microM(-1) s(-1)). The substrate binding of Abz-LFK(Dnp)-OH with testis ACE using a combination of conformational analysis and molecular docking was examined, and the results shed new light on the binding characteristics of the enzyme.     

Fluorescence polarization studies of different forms of angiotensin-converting enzyme

The interaction of three forms of bovine angiotensin-converting enzyme (ACE) with the competitive peptide inhibitor lisinopril with a fluorescent label was studied by the fluorescence polarization technique. The dissociation constants K _d of the enzyme-inhibitor complexes in 50 mM Hepes-buffer, pH 7.5, containing 150 mM NaCl and 1 M ZnCl₂ at 37°C were (2.3 ± 0.4)·10^–8, (2.1 ± 0.3)·10^–8, and (2.1 ± 0.2)·10^–8 M for two-domain somatic ACE, single-domain testicular ACE, and for the N-domain of the enzyme, respectively. The interaction of the enzyme with the inhibitor strongly depended on the presence of chloride in the medium, and the apparent dissociation constant of the ACE-chloride complex was (1.3 ± 0.2)·10^–3 M for the somatic enzyme. The dissociation kinetics of the complex of the inhibitor with somatic ACE did not fit the kinetics of a first-order reaction, but it was approximated by a model of simultaneous dissociation of two complexes with the dissociation rate constants (0.13 ± 0.01) sec^–1 and (0.026 ± 0.001) sec^–1 that were present at approximately equal initial concentrations. The dissociation kinetics of the single-domain ACE complexes with the inhibitor were apparently first-order, and the dissociation rate constants were similar: (0.055 ± 0.001) and (0.041 ± 0.001) sec^–1 for the N-domain and for testicular ACE, respectively.     

Arg(1098) is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity.

Angiotensin (Ang) I-converting enzyme (ACE) is a Zn(2+) metalloprotease with two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I is increased by Cl(-), but the molecular mechanism of this regulation is unclear. A search for single substitutions to Gln among all conserved basic residues (Lys/Arg) in human ACE C-domain identified R1098Q as the sole mutant that lacked Cl(-) dependence. Cl(-) dependence is also lost when the equivalent Arg in the N-domain, Arg(500), is substituted with Gln. The Arg(1098) to Lys substitution reduced Cl(-) binding affinity by approximately 100-fold. In the absence of Cl(-), substrate binding affinity (1/K(m)) of and catalytic efficiency (k(cat)/K(m)) for Ang I hydrolysis are increased 6.9- and 32-fold, respectively, by the Arg(1098) to Gln substitution, and are similar (<2-fold difference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl(-)]. The Arg(1098) to Gln substitution also eliminates Cl(-) dependence for hydrolysis of tetrapeptide substrates, but activity toward these substrates is similar to that of the wild-type C-domain in the absence of Cl(-). These findings indicate that: 1) Arg(1098) is a critical residue of the C-domain Cl(-)-binding site and 2) a basic side chain is necessary for Cl(-) dependence. For tetrapeptide substrates, the inability of R1098Q to recreate the high affinity state generated by the Cl(-)-C-domain interaction suggests that substrate interactions with the enzyme-bound Cl(-) are much more important for the hydrolysis of short substrates than for Ang I. Since Cl(-) concentrations are saturating under physiological conditions and Arg(1098) is not critical for Ang I hydrolysis, we speculate that the evolutionary pressure for the maintenance of the Cl(-)-binding site is its ability to allow cleavage of short cognate peptide substrates at high catalytic efficiencies.     

Insight into the interactive residues between two domains of human somatic Angiotensin-converting enzyme and Angiotensin II by MM-PBSA calculation and steered molecular dynamics simulation

Angiotensin-converting enzyme (ACE), a membrane-bound zinc metallopeptidase, catalyzes the formation of Angiotensin-II (AngII) and the deactivation of bradykinin in the renin–angiotensin-aldosterone and kallikrein–kinin systems. As a hydrolysis product of ACE, AngII is regarded as an inhibitor and displays stronger competitive inhibition in the C-domain than the N-domain of ACE. However, the AngII binding differences between the two domains and the mechanisms behind AngII dissociation from the C-domain are rarely explored. In this work, molecular docking, Molecular Mechanics/Poisson–Boltzmann Surface Area calculation, and steered molecular dynamics (SMD) are applied to explore the structures and interactions in the binding or unbinding of AngII with the two domains of human somatic ACE. Calculated free energy values suggest that the C-domain–AngII complex is more stable than the N-domain–AngII complex, consistent with available experimental data. SMD simulation results imply that electrostatic interaction is dominant in the dissociation of AngII from the C-domain. Moreover, Gln106, Asp121, Glu123, and Tyr213 may be the key residues in the unbinding pathway of AngII. The simulation results in our work provide insights into the interactions between the two domains of ACE and its natural peptide inhibitor AngII at a molecular level. Moreover, the results provide theoretical clues for the design of new inhibitors.     

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE.     

Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.     

Structure and characterization of RNase H3 from Aquifex aeolicus

The crystal structure of ribonuclease?H3 from Aquifex?aeolicus (Aae-RNase?H3) was determined at 2.0?? resolution. Aae-RNase?H3 consists of an N-terminal TATA box-binding protein (TBP)-like domain (N-domain) and a C-terminal RNase?H domain (C-domain). The structure of the C-domain highly resembles that of Bacillus?stearothermophilus RNase?H3 (Bst-RNase?H3), except that it contains three disulfide bonds, and the fourth conserved glutamate residue of the Asp-Glu-Asp-Glu active site motif (Glu198) is located far from the active site. These disulfide bonds were shown to contribute to hyper-stabilization of the protein. Non-conserved Glu194 was identified as the fourth active site residue. The structure of the N-domain without the C-domain also highly resembles that of Bst-RNase?H3. However, the arrangement of the N-domain relative to the C-domain greatly varies for these proteins because of the difference in the linker size between the domains. The linker of Bst-RNase?H3 is relatively long and flexible, while that of Aae-RNase?H3 is short and assumes a helix formation. Biochemical characterizations of Aae-RNase?H3 and its derivatives without the N- or C-domain or with a mutation in the N-domain indicate that the N-domain of Aae-RNase?H3 is important for substrate binding, and uses the flat surface of the β-sheet for substrate binding. However, this surface is located far from the active site and on the opposite side to the active site. We propose that the N-domain of Aae-RNase?H3 is required for initial contact with the substrate. The resulting complex may be rearranged such that only the C-domain forms a complex with the substrate.     

QM/MM investigation of the catalytic mechanism of angiotensin-converting enzyme

Angiotensin-converting enzyme (ACE) converts angiotensin I to angiotensin II and degrades bradykinin and other vasoactive peptides. ACE inhibitors are used to treat diseases such as hypertension and heart failure. It is thus highly desirable to understand the catalytic mechanism of ACE, as this should facilitate the design of more powerful and selective ACE inhibitors. ACE exhibits two different active domains, the C-domain and the N-domain. In this work, we systematically investigated the inhibitor- and substrate-binding patterns in the N-domain of human ACE using a combined quantum mechanical and molecular mechanical approach. The hydrolysis of hippuryl–histidyl–leucine (HHL) as catalyzed by the N-domain of human somatic ACE was explored, and the effects of chloride ion on the overall reaction were also investigated. Two models, one with and one without a chloride ion at the first binding position, were then designed to examine the chloride dependence of inhibitor–substrate binding and the catalytic mechanism. Our calculations indicate that the hydrolysis reaction follows a stepwise general base/general acid catalysis path. The estimated mean free energy barrier height in the two models is about 15.6 kcal/mol, which agrees very well with the experimentally estimated value of 15.8 kcal/mol. Our simulations thus suggest that the N-domain is in a mixed form during ACE-catalyzed hydrolysis, with the single-chloride-ion and the double-chloride-ion forms existing simultaneously.

Open image in new window

Graphical Abstract Superposition of ACE C- and N- domains

     

Angiotensin-converting enzyme: zinc- and inhibitor-binding stoichiometries of the somatic and testis isozymes.

The blood pressure regulating somatic isozyme of angiotensin-converting enzyme (ACE) consists of two homologous, tandem domains each containing a putative metal-binding motif (HEXXH), while the testis isozyme consists of just a single domain that is identical with the C-terminal half of somatic ACE. Previous metal analyses of somatic ACE have indicated a zinc stoichiometry of 1 mol of Zn2+/mol of ACE and inhibitor-binding studies have found 1 mol of inhibitor bound/mol of enzyme. These and other data have indicated that only one of the two domains of somatic ACE is catalytically active. We have repeated the metal and inhibitor-binding analyses of ACE from various sources and have determined protein concentration by quantitative amino acid analysis on the basis of accurate polypeptide molecular weights that are now available. We find that the somatic isozyme in fact contains 2 mol of Zn2+ and binds 2 mol of lisinopril (an ACE inhibitor) per mol of enzyme, whereas the testis isozyme contains 1 mol of Zn2+ and binds 1 mol of lisinopril. In the case of somatic ACE, the second equivalent of inhibitor binds to a second zinc-containing site as evidenced by the ability of a moderate excess of inhibitor to protect both zinc ions against dissociation. However, active site titration with lisinopril assayed by hydrolysis of furanacryloyl-Phe-Gly-Gly revealed that 1 mol of inhibitor/mol of enzyme abolished the activity of either isozyme, indicating that the principal angiotensin-converting site likely resides in the C-terminal (testicular) domain of somatic ACE and that binding of inhibitor to this site is stronger than to the second site.(ABSTRACT TRUNCATED AT 250 WORDS)