Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning.

Abstract

The amino-terminal amino acid sequence and several internal peptide sequences of angiotensin I-converting enzyme (ACE; peptidyl-dipeptidase A, kininase II; EC 3.4.15. 1) purified from human kidney were used to design oligonucleotide probes. The nucleotide sequence of ACE mRNA was determined by molecular cloning of the DNA complementary to the human vascular endothelial cell ACE mRNA. The complete amino acid sequence deduced from the cDNA contains 1306 residues, beginning with a signal peptide of 29 amino acids. A highly hydrophobic sequence located near the carboxylterminal extremity of the molecule most likely constitutes the anchor to the plasma membrane. The sequence of ACE reveals a high degree of internal homology between two large domains, suggesting that the molecule resulted from a gene duplication. Each of these two domains contains short amino acid sequences identical to those located around critical residues of the active site of other metallopeptidases (thermolysin, neutral endopeptidase, and collagenase) and therefore bears a putative active site. Since earlier experiments suggested that a single Zn atom was bound per molecule of ACE, only one of the two domains should be catalytically active. The results of genomic DNA analysis with the cDNA probe are consistent with the presence of a single gene for ACE in the haploid human genome. Whereas the ACE gene is transcribed as a 4.3-kilobase mRNA in vascular endothelial cells, a 3.0-kilobase transcript was detected in the testis, where a shorter form of ACE is synthesized.Peptidyl-dipeptidase A (EC 3.4.15.1) plays an important role in blood pressure homeostasis by hydrolyzing angiotensin I, the inactive peptide released after cleavage of angiotensin by renin, into angiotensin II (1). Accordingly, this Zn metallopeptidase is designated angiotensin I-converting enzyme (ACE), although being the same enzyme as kininase II, it is also able to hydrolyze bradykinin and various other peptides (2, 3). This enzyme is a widely distributed peptidase, occurring, for example, as a membrane-bound ectoenzyme on the surface of vascular endothelial cells and renal epithelial cells and as a circulating enzyme in plasma (3-5). We report here the amino acid sequence of ACE as deduced from the nucleotide sequence of DNA complementary to the ACE mRNA.t MATERIALS AND METHODSPurification and Sequencing of ACE and Preparation of Oligodeoxyribonucleotide Probe. The cortex offresh postmortem human kidneys (600 g) was homogenized (54:100, wt/vol) in 20 mM potassium phosphate buffer (pH 8) containing 250 mM sucrose and a mixture of protease inhibitors, cells debris was discarded, and the particulate fraction was sedimented by centrifugation at 105,000 x g for 1 hr. The pellet was resuspended in 200 ml of 150 mM potassium phosphate buffer (pH 8; buffer I) and treated for 18 hr with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, 8 mM; Serva). The supernatant obtained after centrifugation at 105,000 x g for 1 hr was dialyzed extensively against b...

Neutral endopeptidase and angiotensin I converting enzyme insertion/deletion gene polymorphism in humans

“…In humans, the I/D ACE gene polymorphism is due to the presence (insertion, I) or absence (deletion, D) of a 287 bp sequence in the intron 16 on the chromosome 17q23 6 and determines plasma ACE levels by 40%. The D allele is associated with increased ACE activity.…”

Porcine pulmonary angiotensin I-converting enzyme—Biochemical characterization and spatial arrangement of the N- and C-domains by three-dimensional electron microscopic reconstruction

“…The results are shown in Table 4. The sequence data correspond to residues 1-24 and 616-640 of human sACE (Soubrier et al, 1988) and suggest that they were derived from the N-and Cdomains, respectively. Peak I ( fig.…”

“…This length is long enough to bridge the interdomain distance of 2.0-2.5 nm observed in the electron microscopic density model. Table 4) the tripeptide Leu-Val-Thr (residues 613-615 of human sACE, Soubrier et al, 1988) has to be added to the 'interdomain-linker' of the intact molecule, thus giving it an overall length of 15 amino acids.…”

“…The first full-length cDNA clone for ACE was constructed from the transcripts of ACE gene of human kidney (Soubrier et al, 1988). Human endothelial ACE consists of a single polypeptide chain composed of 1277 residues, of which 14 are cysteine residues.…”

“…The overall sequence similarity between the two domains is 60% in both nucleotide and amino acid sequences but increases to 89% in a sequence of 40 amino acids in each domain containing essential active site residues. Therefore, each domain of sACE contains a catalytic site (Soubrier et al, 1988;Wei et al, 1991). There is no sequence similarity between the carboxyl-and amino-terminal extremities of the sACE molecule, and the central region which separates the two homologous domains, called the 'interdomain linker' (Corradi et al, 2006) is also unique.…”