Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Phosphoramidon, a potent inhibitor of endopeptidase-24.11 (E-24.11) and thermolysin, has been shown to reduce the hypertensive effect of exogenous big endothelin-1 (big ET-1) in rats. To examine whether E-24.11 or thermolysin convert big ET-1 to endothelin-1 (ET-1) and C-terminal fragment (CTF), the effects on porcine and human big ET-1 of each of the purified enzymes were compared in vitro. 2. For E-24.11, the relative rates of hydrolysis were ET-1 > CTF >> big ET-1. The relative half-lives for hydrolysis of 3 nmol of each peptide by 200 ng enzyme were: big ET-1 > 24 h; ET-1, 37 min; CTF, 57 min. For comparison, the half-life for hydrolysis of substance P under similar conditions was 2.1 min. 3. For thermolysin the relative rates of hydrolysis were found to be big ET-1 > CTF > ET-1. The relative half-lives for hydrolysis of 3 nmol peptide by 50 ng enzyme were: big ET-1, 25 min; ET-1, 56 min; CTF, 47 min. 4. Because the low rate of conversion of big ET-1 to ET-1 by E-24.11 did not yield sufficient ET-1 for h.p.l.c. quantification a RIA specific for ET-1(16-21) was used to study further the hydrolysis of big ET-1 by E-24.11. Incubation of big ET-1 (0.2-2 nmol) with E-24.11 (4-400 ng) generated ET-1 levels of between 1.7 and 33 pmol measured by RIA. Incubation of big ET-1 (2 nmol) with E-24.11 (40 ng) for 8 h showed that steady state levels of ET-1 were achieved after 4 h indicating that the rate of ET-1 degradation was then equal to the formation of new ET-1. Characterization of the immunoreactivity by h.p.l.c. and RIA confirmed that authentic ET-1 had been produced, but the yield was insufficient for verification by mass spectrometry.5. Both ET-l-like and CTF-like peaks were detected at 214 nm when the products of big ET-1 hydrolysis by thermolysin were resolved by h.p.l.c. RIA and mass spectrometry confirmed the production of ET-1 with amounts in the range 120-160 pmol.6. The hydrolysis profile of ET-1 by E-24.11 and thermolysin shows that both enzymes have some common cleavage sites consistent with their similar specificities hydrolysing on the amino side of a hydrophobic residue.7. Thermolysin, for which 3D structural information is available, may represent a better model for endothelin converting enzyme (ECE) action than E-24.11 and could be useful for the design of ECE inhibitors. Since E-24.11 can both synthesize and hydrolyse ET-1, the presence of E-24.11 in membrane fractions or in partially purified ECE preparations may produce misleading estimates of ECE activity.
 ...
PMID:Generation by the phosphoramidon-sensitive peptidases, endopeptidase-24.11 and thermolysin, of endothelin-1 and c-terminal fragment from big endothelin-1. 752 8

Hydroxamic acids 6a-h, derived from malonyl amino acids, and 25a-d, derived from succinyl amino acids, were synthesized as inhibitors of human bronchiolar smooth muscle endothelin-converting enzyme (HBSM ECE). Several unexpected side reactions were discovered, particularly in the synthesis of hydroxamates derived from succinates. In vitro evaluation against human bronchiolar ECE revealed that in all cases hydroxamates derived from malonate were more potent than hydroxamates derived from succinate. Isopropyl and isobutyl P1' side chains were suitable; omission of the P1' side chain seriously diminished potency. In the P2' position, several amino acids gave potent malonate-derived hydroxamate inhibitors (6b, d-h, IC50 = 0.2-6.8 nM), and beta-Ala provided an extremely potent inhibitor (6c, IC50 = 0.01 nM). C-terminus carboxylates are much more potent ECE inhibitors than the corresponding amides. Most of the hydroxamates were also potent inhibitors of thermolysin and neutral endopeptidase (NEP); however, the P2' beta-Ala derivative 6c uniquely inhibited HBSM ECE much more potently than NEP.
 ...
PMID:Hydroxamic acids as potent inhibitors of endothelin-converting enzyme from human bronchiolar smooth muscle. 778 43

The structure activity relationship of phosphoramidon analogues was studied for their ability to reduce the hypertensive effect of exogenous proET-1, probably via inhibition of an endothelin converting enzyme activity (ECE). Results concerning in vivo ECE and in vitro thermolysin inhibitions were compared. In contrast to the phosphoryl group of phosphoramidon, which was found to be an absolute requirement, the rhamnose moiety was of very little importance for the inhibition of either enzyme. Furthermore, the tryptophan residue of phosphoramidon appeared to be particularly important for the ECE inhibition, whereas thermolysin inhibition seemed to depend greatly on the leucine residue. It is concluded that in vivo ECE and thermolysin differ in the way they recognise phosphoramidon. The existence of an hydrophobic pocket, specific for the recognition of the tryptophan residue of phosphoramidon, could be proposed for ECE.
 ...
PMID:Structure activity relationship of phosphoramidon derivatives for in vivo endothelin-converting-enzyme inhibition. 802 Aug 72

Arterial tone and water-electrolyte homeostasis are regulated by several peptides, including angiotensin II (AII), bradykinin (BK), atrial natriuretic peptide (ANP) and endothelins (ETs). Changing the concentrations of these peptides in the plasma, tissue, or urine by decreasing the levels of angiotensin II and endothelins and increasing BK and ANP concentrations, is one way of modulating the hemodynamic load. The metabolism of these peptides in essentially controlled by three enzymes, angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP), and endothelin converting enzyme (ECE), which all belong to the group of zinc metallopeptidases. Inhibition of these peptidases by a single compound (a dual inhibitor) that inhibits at once angiotensin II formation and BK and ANP inactivation, causes vasodilatation with reduction in blood pressure with reduction in blood pressure and increases natriuresis. The design of these inhibitors has often be relied on structure-activity studies, based on active-site models derived from structural data on thermolysin (TLN). The results of a large number of pharmacological experiments and those issued from some clinical studies using selective or mixed inhibitors show that in spontaneously hypertensive rats, dual ACE/NEP inhibitors such as S21,402 produce dose-related decreases (-15 to -40 mmHg) in mean arterial pressure and reductions in left ventricular hypertrophy and cardiac size. These compounds produce also an increase in urinary levels of BK, ANP and cGMP associated with enhanced urine output and sodium excretion. Moreover inhibition of NEP appears to improve the cardio- and reno-protective effects resulting from ACE inhibition and could also reduce hypertrophy of vascular walls. Inhibition of ECE seems to result in a weak reduction in blood pressure, an effect which could be emphasized by using dual ECE/ACE or ECE/NEP inhibitors. According to these results mixed dual inhibitors could be of great interest for the treatment of severe hypertension and chronic heart failure. Potent triple inhibitors blocking ACE, NEP and ECE could also be developed.
 ...
PMID:Cell surface metallopeptidases involved in blood pressure regulation: structure, inhibition and clinical perspectives. 976 15

The zinc metalloendopeptidase, thermolysin (EC 3.4.24.27) produced by Bacillus thermoproteolyticus serves as a model of important physiological enzymes such as neprilysin, angiotensin converting enzyme and endothelin converting enzyme. Thermolysin is synthesised as a pre-proenzyme, with an N-terminal prosequence of 204 residues and a mature sequence of 316 residues. The prosequence facilitates the folding of the denatured mature sequence in vitro and the cleavage of the peptide bond linking the pro and mature sequences occurs by an autocatalytic, intramolecular process. With the aim to study the role of the prosequence in vivo and to produce active mutants for structural studies, the mature sequence of thermolysin has now been expressed in Escherichia coli, either alone or with the prosequence as an independent polypeptide, i.e. in trans form. In addition, the mature sequence of an inactive mutant in which Glu143 involved in the catalytic process was replaced by Ala has also been expressed in trans with the prosequence. The results show that the pro-sequence is required to obtain active thermolysin and that a covalent link with the mature sequence is not necessary for the correct folding of the protease in vivo. Moreover, when expressed in E. coli (in trans with the prosequence), the yield of correctly folded E143A mutant was similar to that of the wild-type protease, whereas no mature enzyme was detected when it was expressed as a pre-proenzyme in Bacillus subtilis. These results demonstrate that the thermolysin prosequence acts as an intramolecular chaperone in vivo and open the way to structural studies of catalytic site mutants produced in large quantities in E. coli.
 ...
PMID:The prosequence of thermolysin acts as an intramolecular chaperone when expressed in trans with the mature sequence in Escherichia coli. 992 74

Mammalian endothelin-converting enzyme is a membrane-bound metalloprotease; its C-terminal domain contains sequence motifs characteristic of zinc metalloproteases. We examined residues expected from molecular modelling to be important for substrate binding using selectively mutated recombinant rat ECE-1alpha expressed in CHO cells. A conserved N-A-Ar-Ar (Ar = aromatic) motif is likely to be important for substrate binding. Mutating N550 to Gln or Y552 to Phe reduces Vmax/Km by 8- and 18-fold, respectively. The equivalent residue to Y553 in thermolysin binds the inhibitor through its NH group. Removing this putative interaction by mutating Tyr to Pro destroys activity, but mutating it to Ala or Phe also removes most activity. Mutating G583 (in a conserved GGI motif N-terminal of the zinc-binding helix) to Ala has no measurable effect, but mutating G584 to Ala destroys activity. Changing V583 in the zinc-binding helix to Met, to mimic the sequence pattern in bovine ECE-2, increases Vmax/Km to 1.7-fold that of the wild-type. Assays of phosphoramidon binding follow the pattern of those of substrate binding, but the IC50 of the more potent ECE inhibitor CGS 26303 was not significantly altered by any of these mutations, suggesting that this compound may bind to ECE in a different mode from phosphoramidon.
 ...
PMID:Molecular modelling and site-directed mutagenesis of the active site of endothelin-converting enzyme. 993 Jun 73

Solid-phase synthesis was used to prepare a series of modifications to the selective and potent inhibitor of endopeptidase EC 3.4.24.15 (EP24.15), N-[1(R, S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), which is degraded at the Ala-Tyr bond, thus severely limiting its utility in vivo. Reducing the amide bond between the Ala and Tyr decreased the potency of the inhibitor to 1/1000. However, the replacement of the second alanine residue immediately adjacent to the tyrosine with alpha-aminoisobutyric acid gave a compound (JA-2) that was equipotent with cFP, with a K(i) of 23 nM. Like cFP, JA-2 inhibited the closely related endopeptidase EC 3.4.24.16 1/20 to 1/30 as potently as it did EP24.15, and did not inhibit the other thermolysin-like endopeptidases angiotensin-converting enzyme, endothelin-converting enzyme and neutral endopeptidase. The biological stability of JA-2 was investigated by incubation with a number of membrane and soluble sheep tissue extracts. In contrast with cFP, JA-2 remained intact after 48 h of incubation with all tissues examined. Further modifications to the JA-2 compound failed to improve the potency of this inhibitor. Hence JA-2 is a potent, EP24.15-preferential and biologically stable inhibitor, therefore providing a valuable tool for further assessing the biological functions of EP24.15.
 ...
PMID:Development and characterization of novel potent and stable inhibitors of endopeptidase EC 3.4.24.15. 1062 May 12