The two homologous domains of human angiotensin I-converting enzyme interact differently with competitive inhibitors.

The endothelial angiotensin I-converting enzyme (ACE; EC 3.4.15.1) has recently been shown to contain two large homologous domains (called here the N and C domains), each being a zinc-dependent dipeptidyl carboxypeptidase. To further characterize the two active sites of ACE, we have investigated their interaction with four competitive ACE inhibitors, which are all potent antihypertensive drugs. The binding of [3H] trandolaprilat to the two active sites was examined using the wild-type ACE and four ACE mutants each containing only one intact domain, the other domain being either deleted or inactivated by point mutation of the zinc-coordinating histidines. In contrast with all the previous studies, which suggested the presence of a single high affinity inhibitor binding site in ACE, the present study shows that both the N and C domains of ACE contain a high affinity inhibitor binding site (KD = 3 and 1 X 10(-10) M, respectively, at pH 7.5, 4 degrees C, and 100 mM NaCl). Chloride stabilizes the enzyme-inhibitor complex for each domain primarily by slowing its dissociation rate, as the k-1 values of the N and C domains are markedly decreased (about 30- and 1100-fold, respectively) by 300 mM NaCl. At high chloride concentrations, the chloride effect is much greater for the C domain than for the N domain resulting in a higher affinity of this inhibitor for the C domain. In addition, the inhibitory potency of captopril (C), enalaprilat (E), and lisinopril (L) for each domain was assayed by hydrolysis of Hip-His-Leu. Their Ki values for the two domains are all within the nanomolar range, indicating that they are all highly potent inhibitors for both domains. However, their relative potencies are different for the C domain (L greater than E greater than C) and the N domain (C greater than E greater than L). The different inhibitor binding properties of the two domains observed in the present study provide strong evidence for the presence of structural differences between the two active sites of ACE.     

Angiotensin converting enzyme (kininase II). Molecular and physiological aspects]

The angiotensin I-converting enzyme (kininase II, ECA) is a membrane bound enzyme anchored to the cell membrane through a single transmembrane domain located near its carboxyterminal extremity. Secretion of ACE by the cell occurs most likely as a result of a posttranslational cleavage of the membrane anchor and intracellular region. The ACE molecule is organized into two large highly homologous domains, each bearing consensus sequences for zinc binding in metallopeptidases. Site directed mutagenesis allowed to establish that both domains bear in fact a functional active site, able to convert angiotensin I into angiotensin II and to hydrolyze bradykinin or substance P. The two active sites of ACE, however, do not display the same sensitivity to anion activation (the C terminal active site being more chloride activatable) and also differs in kinetic parameters for peptide hydrolysis. The C terminal active site can hydrolyze faster angiotensin I and substance P and the N terminal active site is able to perform a peculiar endoproteolytic cleavage of an in vitro substrate of ACE, the luteinizing hormone releasing hormone. Both active sites bind with a high affinity, competitive inhibitors but the Kd of the reaction can vary up to 10 between the two active sites. All together, these observations suggest that ACE contains two active sites, whose structure is not exactly identical. They may have a different substrate specificity, however this remains speculative at the present time. Concerning the regulation of ACE gene expression in man, population studies indicated that the large interindividual variability in plasma ACE levels is genetically determined. An insertion/deletion polymorphism located in an intron of ACE gene is associated with differences in the level of ACE in plasma and cells. The physiological and clinical implications of these observations is discussed.     

Probing the basis of domain-dependent inhibition using novel ketone inhibitors of Angiotensin-converting enzyme

Human angiotensin-converting enzyme (ACE) has two homologous domains, the N and C domains, with differing substrate preferences. X-ray crystal structures of the C and N domains complexed with various inhibitors have allowed identification of active site residues that might be important for the molecular basis of this selectivity. However, it is unclear to what extent the different residues contribute to substrate domain selectivity. Here, cocrystal structures of human testis ACE, equivalent to the C domain, have been determined with two novel C domain-selective ketomethylene inhibitors, (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-tryptophan (kAW) and (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-phenylalanine (kAF). The ketone groups of both inhibitors bind to the zinc ion as a hydrated geminal diolate, demonstrating the ability of the active site to catalyze the formation of the transition state. Moreover, active site residues involved in inhibitor binding have been mutated to their N domain counterparts, and the effect of the mutations on inhibitor binding has been determined. The C domain selectivity of these inhibitors was found to result from interactions between bulky hydrophobic side chain moieties and C domain-specific residues F391, V518, E376, and V380 (numbering of testis ACE). Mutation of these residues decreased the affinity for the inhibitors 4-20-fold. T282, V379, E403, D453, and S516 did not contribute individually to C domain-selective inhibitor binding. Further domain-selective inhibitor design should focus on increasing both the affinity and selectivity of the side chain moieties.     

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.     

Crystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design

Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors.     

Characterization of the first angiotensin-converting like enzyme in bacteria: Ancestor ACE is already active

Angiotensin-converting enzyme (ACE) is a metallopeptidase that converts angiotensin I into angiotensin II. ACE is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two active sites, have been characterized. So far, only soluble, single domain ACEs from invertebrates have been cloned, and these have been implicated in reproduction in insects. Furthermore, an ACE-related carboxypeptidase was recently characterized in Leishmania, a unicellular eukaryote, suggesting the existence of ACE in more distant organisms. Interestingly, in silico databank analysis revealed that bacterial DNA sequences could encode putative ACE-like proteins, strikingly similar to vertebrates' enzymes. To gain more insight into the bacterial enzymes, we cloned the putative ACE from the phytopathogenic bacterium, Xanthomonas axonopodis pv. citri, named XcACE. The 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. In vitro expression and biochemical characterization revealed that XcACE is a functional 72 kDa dipeptidyl-carboxypeptidase. As in mammals, this metalloprotease hydrolyses angiotensin I into angiotensin II. XcACE is sensitive to ACE inhibitors and chloride ions concentration. Variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ACE. XcACE characterization demonstrates that ACE is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution.     

The roles of Glu-327 and His-446 in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by NMR spectroscopic and mutational analyses of the enzyme in the transient phosphohistidine intermediate complex

The bisphosphatase domain derived from the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was studied by 1H-13C HMQC NMR spectroscopy of the histidine C2' and H2' nuclei. The bacterially expressed protein was specifically labeled with 13C at the ring C2' position of the histidines. Each of the seven histidine residues gave rise to a single cross-peak in the HMQC spectra, and these were assigned by use of a series of histidine-to-alanine point mutants. His-304, His-344, and His-469 exhibit 13C and 1H resonances that titrated with pH, while the remaining histidine-associated resonances did not. The 13C and 1H chemical shifts indicate that at neutral pH, His-304 and His-446 are deprotonated, while His-469 is protonated. The pKa of His-344 was determined to be 7.04. The 13C chemical shifts suggest that the deprotonated His-258 exists as the N1' tautomer, while His-392 and His-419 are protonated in the resting, wild-type enzyme. Mutation of the remaining member of the catalytic triad, Glu-327, to alanine in the resting enzyme caused an upfield shift of 1.58 and 1.30 ppm in the 1H and 13C dimensions, respectively, and significant narrowing of the His-258 cross-peak. Mutation of His-446 to alanine produced perturbations of the His-258 cross-peak that were similar to those detected in the E327A mutant. The His-392 resonances were also shifted by the E327A and H446A mutations. These observations strongly suggest that residues His-258, Glu-327, His-392, and His-446 exist within a network of interacting residues that encompasses the catalytic site of the bisphosphatase and includes specific contacts with the C-terminal regulatory region of the enzyme. The specifically 13C-labeled bisphosphatase was monitored during turnover by HMQC spectra acquired from the transient N3' phosphohistidine intermediate complex in the wild-type enzyme, the E327A mutant, and the H446A mutant. These complexes were formed during reaction with the physiological substrate fructose-2, 6-bisphosphate. Upon formation of the phosphohistidine at His-258, the 13C and 1H resonances of this residue were shifted downfield by 1.7 and 0.31 ppm, respectively, in the wild-type enzyme. The upfield shifts of the His-258 resonances in the E327A and H446A mutant resting enzymes were reversed when the phosphohistidine was formed, generating spectra very similar to that of the wild-type enzyme in the intermediate complex. In contrast, the binding of fructose-6-phosphate, the reaction product, to the resting enzyme did not promote significant changes in the histidine-associated resonances in either the wild-type or the mutant enzymes. The interpretation of these data within the context of the X-ray crystal structures of the enzyme is used to define the role of Glu-327 in the catalytic mechanism of the bisphosphatase and to identify His-446 as a putative link in the chain of molecular events that results in activation of the bisphosphatase site by cAMP-dependent phosphorylation of the hepatic bifunctional enzyme.     

Homologous substitution of ACE C-domain regions with N-domain sequences: effect on processing, shedding, and catalytic properties

Angiotensin-converting enzyme (ACE) exists as two isoforms: somatic ACE (sACE), comprised of two homologous N and C domains, and testis ACE (tACE), comprised of the C domain only. The N and C domains are both active, but show differences in substrate and inhibitor specificity. While both isoforms are shed from the cell surface via a sheddase-mediated cleavage, tACE is shed much more efficiently than sACE. To delineate the regions of tACE that are important in catalytic activity, intracellular processing, and regulated ectodomain shedding, regions of the tACE sequence were replaced with the corresponding N-domain sequence. The resultant chimeras C1-163Ndom-ACE, C417-579Ndom-ACE, and C583-623Ndom-ACE were processed to the cell surface of transfected Chinese hamster ovary (CHO) cells, and were cleaved at the identical site as that of tACE. They also showed acquisition of N-domain-like catalytic properties. Homology modelling of the chimeric proteins revealed structural changes in regions required for tACE-specific catalytic activity. In contrast, C164-416Ndom-ACE and C191-214Ndom-ACE demonstrated defective intracellular processing and were neither enzymatically active nor shed. Therefore, critical elements within region D164-V416 and more specifically I191-T214 are required for the processing, cell-surface targeting, and enzyme activity of tACE, and cannot be substituted for by the homologous N-domain sequence.     

The structure of testis angiotensin-converting enzyme in complex with the C domain-specific inhibitor RXPA380

Angiotensin I-converting enzyme (ACE) is central to the regulation of the renin-angiotensin system and is a key therapeutic target for combating hypertension and related cardiovascular diseases. Currently available drugs bind both active sites of its two homologous domains, although it is now understood that these domains function differently in vivo. The recently solved crystal structures of both domains (N and C) open the door to new domain-specific inhibitor design, taking advantage of the differences between these two large active sites. Here we present the first crystal structure at a resolution of 2.25 A of testis ACE (identical to the C domain of somatic ACE) with the highly C-domain-specific phosphinic inhibitor, RXPA380. Testis ACE retains the same conformation as seen in previously determined inhibitor complexes, but the RXPA380 central backbone conformation is more similar to that seen for the inhibitor captopril than enalaprilat. The RXPA380 molecule occupies more subsites of the testis ACE active site than the previously determined inhibitors and possesses bulky moieties that extend into the S2' and S2 subsites. Thus the high affinity of RXPA380 for the testis ACE/somatic ACE C domain is explained by the interaction of these bulky moieties with residues unique to these domains, specifically Phe 391, Val 379, and Val 380, that are not found in the N domain. The characterization of the extended active site and the binding of a potent C-domain-selective inhibitor provide the first structural data for the design of truly domain-specific pharmacophores.     

血管紧张素转换酶的结构功能及相关抑制剂

血管紧张素转化酶(angiotensin converting enzyme, ACE, EC 3.4.15.1)是一种位于细胞膜上, 依赖锌离子的羧二肽酶, 催化水解十肽血管紧张素I羧基末端两个氨基酸, 生成具有血管收缩作用的八肽血管紧张素II。ACE在血压调节系统renin - angiotensin system (RAS系统)中具有重要作用, 从ACE的结构功能、基因多态性及其抑制剂等方面进行了详细综述。发现体细胞ACE两个活性中心催化血管紧张素I和缓激肽的机制不同, 因此以体细胞ACE单个活性中心为靶点的研究, 将会为研制开发副作用更少, 安全性更高的ACE抑制剂提供新的途径。     

A catalytic mechanism revealed by the crystal structures of the imidazolonepropionase from Bacillus subtilis

Imidazolonepropionase (EC 3.5.2.7) catalyzes the third step in the universal histidine degradation pathway, hydrolyzing the carbon-nitrogen bonds in 4-imidazolone-5-propionic acid to yield N-formimino-l-glutamic acid. Here we report the crystal structures of the Bacillus subtilis imidazolonepropionase and its complex at 2.0-A resolution with substrate analog imidazole-4-acetic acid sodium (I4AA). The structure of the native enzyme contains two domains, a TIM (triose-phosphate isomerase) barrel domain with two insertions and a small beta-sandwich domain. The TIM barrel domain is quite similar to the members of the alpha/beta barrel metallo-dependent hydrolase superfamily, especially to Escherichia coli cytosine deaminase. A metal ion was found in the central cavity of the TIM barrel and was tightly coordinated to residues His-80, His-82, His-249, Asp-324, and a water molecule. X-ray fluorescence scan analysis confirmed that the bound metal ion was a zinc ion. An acetate ion, 6 A away from the zinc ion, was also found in the potential active site. In the complex structure with I4AA, a substrate analog, I4AA replaced the acetate ion and contacted with Arg-89, Try-102, Tyr-152, His-185, and Glu-252, further defining and confirming the active site. The detailed structural studies allowed us to propose a zinc-activated nucleophilic attack mechanism for the hydrolysis reaction catalyzed by the enzyme.     

Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297.

We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu-297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His-296, His-300 and Glu-319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site-directed mutagenesis experiments were carried out. Glu-297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu-297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.     

Angiotensin converting enzyme: implications from molecular biology for its physiological functions.

1. The two isozymes of human angiotensin converting enzyme (ACE; EC 3.4.15.1) have recently been cloned and sequenced. 2. The larger, endothelial isozyme has two highly similar internal domains each bearing a putative catalytic site. In contrast the smaller, testicular isozyme has a single catalytic site corresponding to the C-terminal domain of endothelial ACE and represents the ancestral, non-duplicated form of the gene. 3. Both isozymes are anchored in the plasma membrane by a single hydrophobic transmembrane polypeptide located near the C-terminus, and both are extensively N-glycosylated. 4. The testicular isozyme may also be O-glycosylated. 5. The soluble form of ACE in plasma, seminal fluid and other body fluids appears to be derived from the membrane-bound endothelial isozyme by a post-translational modification. 6. ACE has a complex substrate specificity with peptidyl tripeptidase or endopeptidase action on certain peptides, as well as the classical peptidyl dipeptidase activity. 7. Numerous potent inhibitors of the enzyme have been developed and used successfully in the treatment of hypertension, but some of the observed side effects may be due to inhibition of other zinc metalloenzymes. 8. Both endothelial and testicular ACE are highly conserved between species, indicative of the essential role(s) of the enzyme in blood pressure regulation and other physiological processes.     

Mixed-Type Inhibition of Pulmonary Angiotensin I-Converting Enzyme by Captopril,Enalaprilat and Ramiprilat

Abstract

We have compared at the enzymological level pulmonary angiotensin I-converting enzymes (ACE) purified to electrophoretic homogeneity from four mammalians species: pig, rat, monkey and human. Using both substrates hippuryl-histidyl-Ieucine and furylacryloyi-phenylal-anyl-glycyi-glycine in steady-state conditions, all the ACES exhibited Michaelis kinetics with identical Michaelis constants, maximal velocities, optimal pH and optimal activating chloride-concentrations. The apparent inhibitory constant was higher for Captopril than for Enalaprilat and even more so for Ramiprilat irrespective of the origin of ACE and the substrate used. Although these inhibitors have been described as competitive inhibitors, Lineweaver-Burk plots were not in accordance with a simple competitive model; moreover, Dixon plots were rather characteristic of non-competitive inhibition. These data emphasize the hypothesis that ACE inhibitors act with mixed-type inhibition, which is consistent with their slow-tight binding to the ACE active center, also with binding of chloride on a critical lysine residue leading to a potential conformational change, and finally with the fact that ACE has two domains, each bearing one catalytic site. On the other hand, as identical kinetic parameters were obtained on the different ACE preparations, results from animal models should allow the extrapolation to humans, in particular for investigations on both renin-angiotensin and kallikrein-kinin systems, and on their inhibition.     

ACEH/ACE2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ACE inhibitors

A human zinc metalloprotease (termed ACEH or ACE2) with considerable homology to angiotensin-converting enzyme (ACE) (EC 3.4.15.1) has been identified and subsequently cloned and functionally expressed. The translated protein contains an N-terminal signal sequence, a single catalytic domain with zinc-binding motif (HEMGH), a transmembrane region, and a small C-terminal cytosolic domain. Unlike somatic ACE, ACEH functions as a carboxypeptidase when acting on angiotensin I and angiotensin II or other peptide substrates. ACEH may function in conjunction with ACE and neprilysin in novel pathways of angiotensin metabolism of physiological significance. In contrast with ACE, ACEH does not hydrolyse bradykinin and is not inhibited by typical ACE inhibitors. ACEH is unique among mammalian carboxypeptidases in containing an HEXXH zinc motif but, in this respect, resembles a bacterial enzyme, Thermus aquaticus (Taq) carboxypeptidase (EC 3.4.17.19). Collectrin, a developmentally regulated renal protein, is homologous with the C-terminal region of ACEH but has no similarity with ACE and no catalytic domain. Thus, the ACEH protein may have evolved as a chimera of a single ACE-like domain and a collectrin domain. The collectrin domain may regulate tissue response to injury whereas the catalytic domain is involved in peptide processing events.     

The M1P1 loop of TASK3 K2P channels apposes the selectivity filter and influences channel function

Channels of the two-pore domain potassium (K2P) family contain two pore domains rather than one and an unusually long pre-pore extracellular linker called the M1P1 loop. The TASK (TASK1, TASK3, and TASK5) subfamily of K2P channels is regulated by a number of different pharmacological and physiological mediators. At pH 7.4 TASK3 channels are selectively blocked by zinc in a manner that is both pH(o)- and [K](o)(-)dependent. Mutation of both the Glu-70 residue in the M1P1 loop and the His-98 residue in the pore region abolished block, suggesting the two residues may contribute to a zinc binding site. Mutation of one Glu-70 residue and one His-98 residue to cysteine in TASK3 fixed concatamer channels gave currents that were enhanced by dithiothreitol and then potently blocked by cadmium, suggesting that spontaneous disulfide bridges could be formed between these two residues. Swapping the M1P1 loops of TASK1 and TASK3 channels showed that the M1P1 loop is also involved in channel regulation by pH. Therefore, the TASK3 M1P1 loop lies close to the pore, regulating TASK3 channel activity.     

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.     

Role of the N-terminal catalytic domain of angiotensin-converting enzyme investigated by targeted inactivation in mice

Angiotensin-converting enzyme (ACE) produces the vasoconstrictor angiotensin II. The ACE protein is composed of two homologous domains, each binding zinc and each independently catalytic. To assess the physiologic significance of the two ACE catalytic domains, we used gene targeting in mice to introduce two point mutations (H395K and H399K) that selectively inactivated the ACE N-terminal catalytic site. This modification does not affect C-terminal enzymatic activity or ACE protein expression. In addition, the testis ACE isozyme is not affected by the mutations. Analysis of homozygous mutant mice (termed ACE 7/7) showed normal plasma levels of angiotensin II but an elevation of plasma and urine N-acetyl-Ser-Asp-Lys-Pro, a peptide suggested to inhibit bone marrow maturation. Despite this, ACE 7/7 mice had blood pressure, renal function, and hematocrit that were indistinguishable from wild-type mice. We also studied compound heterozygous mice in which one ACE allele was null (no ACE expression) and the second allele encoded the mutations selectively inactivating the N-terminal catalytic domain. These mice produced approximately half the normal levels of ACE, with the ACE protein lacking N-terminal catalytic activity. Despite this, the mice have a phenotype indistinguishable from wild-type animals. This study shows that, in vivo, the presence of the C-terminal ACE catalytic domain is sufficient to maintain a functional renin-angiotensin system. It also strongly suggests that the anemia present in ACE null mice is not due to the accumulation of the peptide N-acetyl-Ser-Asp-Lys-Pro.     

Structure-function analysis of Trypanosoma brucei RNA triphosphatase and evidence for a two-metal mechanism

Trypanosoma brucei RNA triphosphatase TbCet1 is a 252-amino acid polypeptide that catalyzes the first step in mRNA cap formation. By performing an alanine scan of TbCet1, we identified six amino acids that are essential for triphosphatase activity (Glu-52, Arg-127, Glu-168, Arg-186, Glu-216, and Glu-218). These results consolidate the proposal that protozoan, fungal, and Chlorella virus RNA triphosphatases belong to a single family of metal-dependent NTP phosphohydrolases with a unique tunnel active site composed of eight beta strands. Limited proteolysis of TbCet1 suggests that the hydrophilic N terminus is surface-exposed, whereas the catalytic core domain is tightly folded with the exception of a protease-sensitive loop (76WKGRRARKT84) between two of the putative tunnel strands. The catalytic domain of TbCet1 is extraordinarily thermostable. It remains active after heating for 2 h at 75 degrees C. Analysis by zonal velocity sedimentation indicates that TbCet1 is a monomeric enzyme, unlike fungal RNA triphosphatases, which are homodimers. We show that tripolyphosphate is a potent competitive inhibitor of TbCet1 (Ki 1.4 microm) that binds more avidly to the active site than the ATP substrate (Km 25 microm). We present evidence of synergistic activation of the TbCet1 triphosphatase by manganese and magnesium, consistent with a two-metal mechanism of catalysis. Our findings provide new insight to the similarities (in active site tertiary structure and catalytic mechanism) and differences (in quaternary structure and thermal stability) among the different branches of the tunnel enzyme family.