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

Endothelin-converting enzyme (ECE) is a member of the zinc metalloproteinase family. It is much more specific in its protease activity than the bacterial metalloprotease thermolysin; we aim to construct a model of its active site to help to explain these differences. We aligned the sequence of human ECE with those of human neprilysin (which is 39% identical to ECE) and thermolysin. Residues believed to be important for inhibitor binding were assigned from the alignment and by analogy with structural and functional studies of these enzymes. These included a conserved IGG motif N-terminal to the zinc-binding HExxH motif, and a tyrosine residue that may be analogous to Y157 of thermolysin. We have used the program O to build a model of the active site of ECE based on the crystal structure of thermolysin.
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PMID:Molecular modeling of the active site of endothelin-converting enzyme. 858 73

A neutral endopeptidase (NEP) from Lactococcus lactis has recently been cloned and shown to contain high sequence homology with the human neutral endopeptidase, endopeptidase 24.11 (I. Mierau et al., J. Bacteriol. 175, 2087-2096, 1993). The gene for the neutral endopeptidase from L. lactis was cloned into the pQE expression vector, resulting in the fusion of a hexahistidine at the N-terminus. The recombinant enzyme was expressed to high levels in Escherichia coli (approximately 10 mg/liter of culture) and purified to homogeneity in a two-step procedure. A number of peptides were studied as substrates for the enzyme. The enzyme cleaves the following peptides at the Gly3-Phe4 bond: enkephalins, dynorphins A-6, A-8, A-9, A-10, A-13, and A-17, and alpha-neo-endorphin. In addition the enzyme hydrolyzes bradykinin, substance P, beta-endorphin, ACTH, and VIP. Although the cleavage patterns observed are similar to that seen with mammalian neutral endopeptidase, the lactococcal enzyme more efficiently cleaves larger peptide substrates. As observed with the mammalian neutral endopeptidase, the lactococcal enzyme exhibits higher kcat/K(m) values for the enkephalins than for their corresponding amides, indicating the functionality of an active-site arginine. Inactivation of the lactococcal endopeptidase by diethyl pyrocarbonate and protection afforded by the substrate dynorphin A-6 indicate the functionality of a positionally conserved active-site histidine. This was confirmed by demonstrating that conversion of this histidine, histidine 587, to glutamine generated inactive enzyme. Similarly, conversion of the putative zinc ligand glutamate 535 to glutamine led to inactive enzyme. These studies indicate a conservation of critical catalytic residues between the two enzymes and suggest that the lactococcal endopeptidase is a better model than thermolysin for the mammalian enzyme.
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PMID:Heterologous expression and characterization of recombinant Lactococcus lactis neutral endopeptidase (neprilysin). 880 62

Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds with the carbonyl group of a residue in the P1 position and also participates in a hydrogen bond network involving Asp-170. Sequence alignment data shows that Arg-717 of neprilysin could play a similar role to Arg-203 of thermolysin. This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. This led, in both cases, to decreases in kcat/Km values, of 122-fold for neprilysin and 2300-fold for thermolysin, essentially due to changes in kcat. The Ki values of several inhibitors were also increased for the mutated enzymes. In addition, the replacement of Asp-170 of thermolysin by Ala residue resulted in a decrease in kcat/Km of 220-fold. The results, coupled with a molecular modeling study, suggest that Arg-717 of neprilysin corresponds to Arg-203 of thermolysin and that in both enzymes a hydrogen bond network exists, involving His-142, Asp-170, and Arg-203 in thermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which is crucial for hydrolytic activity.
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PMID:Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding. 937 73

Two intramolecularly quenched fluorogenic peptides containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-DArg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were K(m) = 2.8 microM, kcat = 5.3 min-1, kcat/K(m) = 2 min-1 microM-1 and K(m) = 5.0 microM, kcat = 7.0 min-1, kcat/K(m) = 1.4 min-1 microM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by alpha-chymotrypsin. The high specificity of these substrates suggests their use for specific NEP assays in crude enzyme preparations.
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PMID:Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases. 949 31

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.
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PMID:Cell surface metallopeptidases involved in blood pressure regulation: structure, inhibition and clinical perspectives. 976 15

Important homologies in the topology of the catalytic site and the mechanism of action of thermolysin and neprilysin have been evidenced by site-directed mutagenesis. The determination of differences in transition state stabilization between these peptidases could facilitate the design of specific inhibitors. Thus, two residues of thermolysin which could be directly (Tyr157) or indirectly (Asp226) involved in the stabilization of the transition state and their putative counterparts in neprilysin (Tyr625 and Asp709) have been mutated. The results show that Tyr157 is important for thermolysin activity while Tyr625 has no functional role in neprilysin. Conversely, the mutation of Asp226 induced a slight perturbation of thermolysin activity while Asp709 in neprilysin seems crucial in neprilysin catalysis. Taken together these data seem to indicate differences in the transition state mode of stabilization in the two peptidases.
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PMID:Differences in transition state stabilization between thermolysin (EC 3.4.24.27) and neprilysin (EC 3.4.24.11). 982 48

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.
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PMID:The prosequence of thermolysin acts as an intramolecular chaperone when expressed in trans with the mature sequence in Escherichia coli. 992 74

A three-dimensional model of the 507-749 region of neutral endopeptidase-24.11 (NEP; E.C.3.4.24.11) was constructed integrating the results of secondary structure predictions and sequence homologies with the bacterial endopeptidase thermolysin. Additional data were extracted from the structure of two other metalloproteases, astacin and stromelysin. The resulting model accounts for the main biological properties of NEP and has been used to describe the environment close to the zinc atom defining the catalytic site. The analysis of several thiol inhibitors, complexed in the model active site, revealed the presence of a large hydrophobic pocket at the S1' subsite level. This is supported by the nature of the constitutive amino acids. The computed energies of bound inhibitors correspond with the relative affinities of the stereoisomers of benzofused macrocycle derivatives of thiorphan. The model could be used to facilitate the design of new NEP inhibitors, as illustrated in the paper.
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PMID:A three-dimensional construction of the active site (region 507-749) of human neutral endopeptidase (EC.3.4.24.11). 1019 85

A novel cDNA, designated human metalloendoprotease 1 (hMP1), was identified on the basis of homology to known metalloendoproteases of the pitrilysin family. The full-length MP1 codes for a protein with an open reading frame of 1038 amino acids. The N-terminal region contains the HXXEH(X)76E catalytic domain that is conserved in the members of pitrilysin family, namely insulin-degrading enzyme and NRD convertase. The hMP1 mRNA is expressed in a number of cell lines and tissues as a single species of about 3.4 kb. The expression of hMP1 mRNA is higher in muscle and heart than in brain, pancreas, liver, lung, and placenta. The full-length hMP1 was expressed in the baculovirus system and purified to homogeneity using isoelectrofocusing and ion-exchange chromatography. The enzyme exhibited a neutral pH optimum and high sensitivity to thiol reagents. HMP1 was inactivated by 1,10-phenanthroline, a specific inhibitor of Zn(+2)-dependent metalloproteases. The enzyme was not inhibited by agents that inhibit neutral metalloendoproteases of the thermolysin family such as thimet endo-oligopeptidase, enkephalinase, or angiotensin-converting enzyme. HMP1 cleaved a prodynorphin-derived peptide, leumorphin, N-terminal to Arg in the monobasic processing site, as evidenced by MALDI-TOF mass spectrometry. However, the enzyme did not exhibit strict monobasic cleavage specificity, as peptide substrates with amino acid substitutions around the monobasic site was cleaved efficiently by hMP1. Taken together, these results suggest that hMP1 is a novel member of the metalloendoprotease superfamily with ubiquitous distribution that could play a broad role in general cellular regulation.
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PMID:Cloning, expression, and characterization of human metalloprotease 1: a novel member of the pitrilysin family of metalloendoproteases. 1036 Aug 38

Three alpha-mercaptoacyldipeptides differing essentially in the size of their C-terminal residues have been crystallized in the thermolysin active site. A new mode of binding was observed for 3 [HS-CH(CH(2)Ph)CO-Phe-Tyr] and 4 [HS-CH((CH(2))(4)CH(3))CO-Phe-Ala], in which the mercaptoacyl moieties act as bidentates with Zn-S and Zn-O distances of 2.3 and 2.4 A, respectively, the side chains fitting the S(1), S(1)', and S(2)' pockets. Moreover, a distance of 3.1 A between the sulfur atom and the OE1 of Glu(143) suggests that they are H-bonded and that one of these atoms is protonated. This H-bond network involving Glu(143), the mercaptoacyl group of the inhibitor, and the Zn ion could be considered a "modified" transition state mimic of the peptide bond hydrolysis. Due to the presence of the hindering (5-phenyl)proline, the inhibitor HS-CH(CH(2)Ph)CO-Gly-(5-Ph)Pro (2) interacts through the usual Zn monodentation via the thiol group and occupancy of S(1)' and S(2)' subsites by the aromatic moieties, the proline ring being outside the active site. The inhibitory potencies are consistent with these structural data, with higher affinities for 3 (4.2 x 10(-)(8) M) and 4 (4.8 x 10(-)(8) M) than for 2 (1.2 x 10(-)(6) M). The extension of the results, obtained with thermolysin being considered as the model of physiological zinc metallopeptidases, allows inhibitor-recognition modes for other peptidases, such as angiotensin converting enzyme and neutral endopeptidase, to be proposed and opens interesting possibilities for the design of new classes of inhibitors.
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PMID:Crystal structures of alpha-mercaptoacyldipeptides in the thermolysin active site: structural parameters for a Zn monodentation or bidentation in metalloendopeptidases. 1050 25


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