Reaction of neutral endopeptidase 24.11 (enkephalinase) with arginine reagents

The effect of the arginine-specific reagents phenylglyoxal and butanedione on the activity of neutral endopeptidase 24.11 ("enkephalinase") was determined. Inactivation of the enzyme by butanedione is completely protected by methionine-enkephalin, but only partially protected by methionine-enkephalinamide. In contrast, phenylglyoxal inactivation of the enzyme exhibits saturation kinetics with a Kd of 20 mM. The enzyme is only partially protected against phenylglyoxal inactivation by both methionine-enkephalin and its amide, indicating that phenylglyoxal reacts at two sites. Reaction of the enzyme with phenylglyoxal in the presence of saturating methionine-enkephalin involves the direct reaction of the reagent with the enzyme-substrate complex. Enzyme treated with butanedione or with phenylglyoxal (at site 1) exhibits a 3-5 decrease in substrate binding with little change in kcat. In contrast, reaction with phenylglyoxal in the presence of saturating methionine-enkephalin shows little change in substrate binding but a 4-fold decrease in kcat. Enzyme inactivation involves the incorporation of approximately 1 mol of phenylglyoxal/enzyme subunit in the absence of methionine-enkephalin and approximately 2.5 mol of phenylglyoxal/enzyme subunit in the presence of saturating methionine-enkephalin. These results suggest that an arginine residue on the enzyme is involved in substrate binding.     

The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase)

Endothelins 1-3 are a family of 21-amino acid peptides whose structure consists of two rings formed by intra-chain disulfide bonds and a linear "COOH-terminal tail." These peptides were originally described on the basis of their potent vasoconstrictor activity. The hydrolytic inactivation of endothelin action has recently been implicated to be attributed, at least in part, to the enzyme neutral endopeptidase 24.11 (Scicli, A. G., Vijayaraghavan, J., Hersh, L., and Carretero, O. (1989) Hypertension 14, 353). The kinetic properties and mode of hydrolysis of the endothelins by this enzyme are reported in this study. The Km for endothelins 1 and 3 hydrolysis is approximately 2 microM while endothelin2 exhibits a 5-fold higher Km. Endothelins 1 and 2 exhibit similar Vmax values while endothelin3 is hydrolyzed considerably more slowly. The initial cleavage site in endothelin1 is at the Ser5-Leu6 bond located within one of the cyclic structures. Thermolysin, a bacterial neutral endopeptidase with a similar substrate specificity to neutral endopeptidase 24.11 initially cleaves endothelin1 between His16-Leu17 which lies within the COOH-terminal linear "tail" portion of the molecule. The cleavage of endothelins 2 and 3 by neutral endopeptidase 24.11 differs from that observed with endothelin1 in that cleavage of these endothelins occurs at Asp18-Ile19 within the linear COOH-terminal tail structure. These results demonstrate that the endothelins are good substrates for neutral endopeptidase 24.11 and suggest that their mode of cleavage is dependent upon both amino acid sequence as well as peptide conformation.     

Fluorogenic substrates for the enkephalin-degrading neutral endopeptidase (Enkephalinase)

Rat brain neutral endopeptidase ("Enkephalinase") was shown to hydrolyze a series of fluorogenic substrates of the general structure 2-aminobenzoyl-(amino acid)n- leucylalanylglycine -4- nitrobenzylamide . The hydrolysis of these substrates was competitively inhibited by Leu5-enkephalin, demonstrating that these are indeed substrates for the rat brain neutral endopeptidase. Cleavage of the fluorogenic substrates yielded leucylalanylglycine -4- nitrobenzylamide as a common product. In addition, a series of inhibitors previously shown to inhibit thermolysin-like enzymes inhibited the hydrolysis of both Leu5-enkephalin and the synthetic substrates. The results of this study (a) demonstrate that the enkephalin-degrading endopeptidase is similar in specificity to thermolysin, (b) provide a continuous sensitive assay system for the enzyme, and (c) point out the potential use of this substrate class for probing the specificity of the enzyme.     

Expression of enzymatically active enkephalinase (neutral endopeptidase) in mammalian cells

A cDNA encoding the rat enkephalinase protein (neutral endopeptidase; EC 3.4.24.11) has been constructed from overlapping lambda gt10 cDNA clones. This cDNA was inserted into an expression plasmid containing the cytomegalovirus enhancer and promoter. When transfected with this plasmid, Cos 7 cells transiently expressed the enkephalinase protein in a membrane-bound state. Recombinant enkephalinase recovered in solubilized extracts from transfected Cos 7 cells was enzymatically active and displayed properties similar to those of the native enzyme with respect to sensitivity to classical enkephalinase inhibitors.     

Comparison of the subsite specificity of the mammalian neutral endopeptidase 24.11 (enkephalinase) to the bacterial neutral endopeptidase thermolysin

A comparison has been made of the specificity of the mammalian neutral metalloendopeptidase, endopeptidase 24.11, with that of the bacterial neutral metalloendopeptidase thermolysin. A series of synthetic oligopeptides which have previously been studied as substrates for thermolysin and used in computer modeling were examined as substrates for the mammalian enzyme. It was found that P1, P2, and P'3 subsite interactions in the mammalian enzyme, although similar to those found in thermolysin, are less restrictive spatially and are considerably less dependent on hydrophobic interactions. This difference was maximally expressed with the synthetic substrate dansyl-D-alanylglycylnitrophenylalanylglycine which is a substrate for the mammalian enzyme, but not for the bacterial enzyme. A comparison of substrates in the free acid form with their corresponding amides showed that binding to the mammalian enzyme is dependent in part on an ionic interaction between the substrate carboxylate group and the enzyme. Such an ionic interaction was not observed with the bacterial enzyme.     

Down-regulation and inactivation of neutral endopeptidase 24.11 (enkephalinase) in human neutrophils

Neutral endopeptidase (EC 3.4.24.11, NEP) is an integral membrane protein of human neutrophils. NEP is identical with the common acute lymphoblastic leukemia antigen (CALLA) of leukemic cells. The expression of NEP on the surface of neutrophils is down-regulated by endocytosis which can be induced by phorbol 12-myristate 13-acetate (PMA) at 37 degrees C. The activity of the enzyme on the surface of intact cells decreases by 76% within 5 min. The activity can be recovered, however, if the cells are lysed within 5 min of the endocytosis. After 30 min, only 32% of the NEP activity is present in the neutrophil lysates. The loss of activity is presumably due to proteolytic inactivation. Diacylglycerol and monoclonal antibody to CALLA/NEP also induce internalization of NEP. PMA induces endocytosis even at 4 degrees C, but NEP is not inactivated at that temperature. The disappearance of NEP activity after adding PMA was inhibited by various agents. Among the most active were the phospholipase inhibitor 4-bromophenacyl bromide and a combination of the serine protease and cathepsin inhibitors, diisopropylfluorophosphate and N-ethylmaleimide. The employment of fluorescent monoclonal antibody confirmed the down-regulation and internalization of NEP antigen on the neutrophils. Since NEP inactivates chemotactic peptides and thereby affects chemotaxis of neutrophils (Painter, R. G., Dukes, R., Sullivan, J., Carter, R., Erd?s, E. G., and Johnson, A. R. (1988) J. Biol. Chem. 263, 9456-9461), the down-regulation of NEP activity on the cell membrane may modulate the function of these cells in inflammation.     

Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase)

We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a lambda gt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N-terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy-terminus, located extracellularly.     

Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis.

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.     

Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA

[This corrects the article on p. 1317 in vol. 6, PMID: 2440677.].     

Mutational analysis reveals only one catalytic histidine in neutral endopeptidase ("enkephalinase").

Aside from serving as zinc ligands, kinetic data has implicated one or more additional histidines as catalytic residues in neutral endopeptidase ("enkephalinase") action. One of these histidines has previously been identified as histidine 704 (Bateman et al., J. Biol. Chem., 265:8365-8368, 1990). In order to determine whether a second histidine is involved in catalysis each of these residues not previously changed have been converted to glutamine by site directed mutagenesis. The resultant recombinant enzymes possess full catalytic activity indicating that histidine 704 is the only catalytic histidine in the enzyme.     

Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pKa is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pKa is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases kappa cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pKa of 7 for NEP and a pKa of 6 for thermolysin. In the case of thermolysin this pKa was decreased to 5.3 in the enzyme-inhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of Ki on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pKa value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pKa of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pKa of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.     

Effect of human endopeptidase 24.11 (enkephalinase) on IL-1-induced thymocyte proliferation activity

Endopeptidase 24.11 (enkephalinase) is a membrane bound protease involved in the degradation of neuropeptides and hormones. Its presence on cells of the thymus and lymph nodes suggests a possible role in the inactivation of immune system mediators. IL-1 (both purified IL-1 beta and an IL-1-rich supernatant) bioactivity, as measured in the thymocyte proliferation assay, was found to disappear upon incubation with endopeptidase 24.11. This inactivation was dependent on both incubation time and enzyme concentration. IL-1 beta was protected by the presence in the incubation medium of phosphoramidon, a specific inhibitor of endopeptidase 24.11. After incubation of IL-1-rich supernatant with the enzyme, the thymocyte proliferation activity could be restored by adding purified IL-1 beta to the samples, indicating that neither the enzyme nor the buffer had any toxic effect on thymocyte proliferation. In the same experimental conditions, IL-2 activity was not destroyed by endopeptidase 24.11.     

Preferential cleavage of amino- and carboxyl-terminal oligopeptides from vasoactive intestinal polypeptide by human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11)

Human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) cleaved synthetic vasoactive intestinal peptide (VIP1-28) with time-and peptidase concentration-dependence, which left less than 30% intact after 30 micrograms was incubated at 37 degrees C with 0.1 micrograms and 10 micrograms of peptidase for 120 min and 15 min, respectively. The rank order of relative rates of peptidolysis amino-terminal to hydrophobic amino acids was Ala4 and Val5 greater than Tyr22 and Ile26 much greater than Leu13 and Met17. The many effects of VIP1-28 on epithelial cell and leukocyte functions thus may be influenced by degradation of the mediator by enkephalinase at the surface of target cells.     

High concentration of neutral endopeptidase (enkephalinase E.C. 3.4.24.11) in a malignant tumor: rat hepatoma 3924A

The activity of the membrane-bound neutral endopeptidase 24.11 was low in the normal liver (21 +/- 3 pmol/h/mg protein, mean +/- SE) but it increased 56-fold in rapidly-growing rat hepatoma 3924A. The identity of the enzyme in the tumor was established by immunoprecipitation and by using a specific inhibitor of neutral endopeptidase. The endopeptidase concentration in the differentiating and regenerating liver was lower than in normal tissue, 39 and 8% of the corresponding control. The activity of a plasma membrane marker enzyme carboxypeptidase M in the normal liver was 1.0 +/- 0.2 nmol/h/mg protein, it increased about 2-fold in the rapidly-growing hepatoma and in the differentiating liver, but was unchanged in regenerating liver. The function of the strikingly increased neutral endopeptidase activity in the rapidly growing hepatoma may relate to activation of autocrine or exocellular growth factors or to inactivation of cell proliferation-inhibitory factors. Such a biochemical change should confer selective advantages to the cancer cells.     

Identification of the active-site arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine 102 and analysis of a glutamine 102 mutant

Neutral endopeptidase 24.11 contains an active-site arginine residue involved in binding the free carboxylate of substrate peptides and inhibitors. This arginine reacts rapidly with [14C]phenylglyoxal, and its reaction is selectively blocked by the presence of either the substrate Met5-enkephalin, the competitive inhibitor phenylalanylalanine, or the transition state analog phosphoramidon. The phenylglyoxal-modified peptide was isolated by a procedure involving limited digestion by trypsin, separation of the tryptic peptides by high pressure liquid chromatography (HPLC), further digestion of the modified peptide by pepsin, and a final purification by HPLC. By this procedure arginine 102 was identified as the active-site arginine. Verification of this finding came from the use of site-directed mutagenesis in which this arginine was replaced by glutamine. Both the mutant and wild-type enzyme reacted equally well with an amide containing substrate, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. However, reaction of the mutant enzyme with a substrate containing a free COOH-terminal carboxylate, 5-dimethylaminonaphthalene-1-sulfonyl-D-Ala-Gly-(NO2)Phe-Gly, was barely detectable with the mutant enzyme. Similarly the mutant enzyme showed a loss of selectivity in inhibition by D-Ala2-Met5-enkephalin compared to the corresponding amide but exhibited no difference in the maximal velocity for hydrolysis of D-Ala2-Met5-enkephalin and its amide.     

Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease

Maturation of the hepatitis C virus (HCV) polyprotein occurs by a series of proteolytic processes catalyzed by host cell proteases and the virally encoded proteases NS2 and NS3. Although several peptidomimetic inhibitors of NS3 protease have been published, only a few small molecule inhibitors have been reported. In an effort to improve screening efficiency by minimizing the spectral interference of various test compounds, a substrate that contains the longer wavelength fluorescence resonance energy transfer (FRET) pair, TAMRA/QSY-7, was devised. For the optimized substrate T-Abu-Q, with sequence Ac-Asp-Glu-Lys(TAMRA)-Glu-Glu-Abu-Psi(COO)Ala-Ser-Lys(QSY-7)-amide, the kinetic parameters with HCV NS3 protease are K(m)=30 microM, k(cat)=0.6s(-1), and k(cat)/K(m)=20,100s(-1)M(-1). We show that this substrate is suitable for inhibitor analysis and mechanistic studies so long as the substrate concentration is low enough (0.5 microM) to avoid complications from high inner filter effects. The substrate is especially useful with ultra-high-density screening formats, such as microarrayed compound screening technology, because there is less spectral interference from the compounds being tested than with more traditional (EDANS/DABCYL) FRET protease substrates. The merits of the new substrate, as well as potential applications of this FRET pair to other protease substrates, are discussed.     

A fluorescence resonance energy transfer sensor based on maltose binding protein

A fluorescence resonance energy-transfer (FRET) sensing system for maltose based on E. coli maltose binding protein (MBP) is demonstrated. The FRET donor portion of the sensing system consists of MBP modified with long wavelength-excitable cyanine dyes (Cy3 or Cy3.5). The novel acceptor portion of the sensor consists of beta-cyclodextrin (beta-CD) modified with either the cyanine dye Cy5 or the dark quencher QSY9. Binding of the modified beta-CD to dye-conjugated MBP results in assembly of the FRET complex. Added maltose displaces the beta-CD-dye adduct and disrupts the FRET complex, resulting in a direct change in fluorescence of the donor moiety. In the use of these FRET pairs, MBP dissociation values for maltose were estimated (0.14-2.90 microM). Maltose limits of detection were in the 50-100 nm range.     

In vitro degradation of endothelin-1 by endopeptidase 24.11 (enkephalinase).

The breakdown of endothelin-1 by crude membrane preparations of human kidney and choroid plexus was investigated. 125I-labeled endothelin-1 was degraded by both tissues in a phosphoramidon-sensitive way, suggesting a role of endopeptidase 24.11 in the in vitro metabolism of this peptide. Identification of the cleavage sites of purified human renal endopeptidase 24.11 in the sequence of endothelin-1 revealed that bonds involving the amino side of the hydrophobic amino acids (Ser4, Leu6, Val12, Phe14, His16, Leu17, Ile19) were susceptible to cleavage. Endothelin-1 appears thus to be degraded at multiple sites by endopeptidase 24.11 in vitro, producing inactive fragments.