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)

The regulatory subunit of cAMP-dependent protein kinase I has been cleaved proteolytically into two structurally independent domains. The larger domain (35K with trypsin or thermolysin and 31K with chymotrypsin) corresponded to the COOH-terminal end of the polypeptide chain and retained the cAMP binding site(s). The smaller domain (11 to 12K with trypsin), corresponding to the NH2-terminal region of the regulatory subunit, contained the region of dimer interaction. In the absence of reducing reagent, the two protomers of the native regulatory subunit and of the smaller domain could be covalently cross-linked by a disulfide bond. In addition to the two major domains, a 15-residue peptide that links the two domains has been isolated and partially characterized. Two major sites on the type I regulatory subunit were susceptible to proteolytic degradation. Site 1, susceptible to cleavage by both trypsin and thermolysin, has the following sequence: LysArg-Arg-Gly-Ala-Ile-Ser-Ala-. Cleavage at this site generated a 35K cAMP-binding fragment. Site 2 contained a chymotryptic cleavage site as well as a secondary tryptic site. The sequence at Site 2 was Val-Arg-Arg-Val-Ile-Ala. Cleavage here generated a 31K cAMP-binding fragment. Both sites contained 2 consecutive basic amino acid residues similar to the corresponding sequence in the type II regulatory subunit; however, in the case of the type I regulatory subunit, the serine at Site 1 does not serve as a site of autophosphorylation. In contrast to the dissociated regulatory subunit, the holoenzyme is partially protected from proteolytic degradation.
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PMID:The structural domains of cAMP-dependent protein kinase I. Characterization of two sites of proteolytic cleavage and homologies to cAMP-dependent protein kinase II. 743 94

A number of structural experimental methods are available to determine the receptor-bound conformation of ligands as part of the process of rational drug design, including X-ray diffraction and solution-state NMR. Not all receptor/ligand systems are amenable to these types of analyses due to difficulties in sample preparation or inherent limitations of the methods. Rotational echo double-resonance (REDOR) NMR is a solid-state, magic angle-spinning technique that measures the dipolar coupling between specifically labeled nuclei and enables the determination of internuclear distance. In previous studies of helical peptides, we have verified the ability of REDOR NMR to measure distances accurately and precisely. In this study we use REDOR and double REDOR to measure distances between backbone atoms in a phosphonamidate transition-state inhibitor bound to thermolysin. The 31P-13C', 31P-15N, and 31P-13C alpha distances (3.61 +/- 0.10, 3.89 +/- 0.12, and 5.37 +/- 0.13 A, respectively) measured in a complex of Cbz-GlyP-[1-13C]Leu-[15N,2-13C]Ala and the enzyme are consistent with those observed by X-ray diffraction in other comparable thermolysin/inhibitor complexes (average values of 3.58 +/- 0.04, 3.91 +/- 0.13, and 5.17 +/- 0.18 A, respectively). These results demonstrate that REDOR NMR is a viable alternative to more traditional methods such as X-ray diffraction, transferred NOESY, and isotope-edited NOESY for characterizing the receptor-bound conformation of ligands.
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PMID:Solid-state nuclear magnetic resonance analysis of the conformation of an inhibitor bound to thermolysin. 762 12

The pre-steady state process in the thermolysin-catalyzed hydrolysis of Cbz-Gly-Phe-Ala was observed at pH 4.5 by fluorescence stopped-flow method using Dns-Phe as a displacement probe. After the confirmation of the pre-equilibrium hypothesis for the binary interaction, the nonlinear substrate concentration dependence of the apparent kinetic constant for the pre-steady state process was analyzed and an existence of multi-intermediates was proposed.
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PMID:Observation of the pre-steady state process in thermolysin catalysis with a fluorescent displacement probe at low pH. 772 Aug 70

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases involved in tissue remodeling. They have also been implicated in various disease processes including tumour invasion and joint destruction and are therefore attractive targets for inhibitor design. For rational drug design, information of inhibitor binding at the atomic level is essential. Recently, we have published the refined high-resolution crystal structure of the catalytic domain of human neutrophil collagenase (HNC) complexed with the inhibitor Pro-Leu-Gly-NHOH, which is a mimic for the unprimed (P3-P1) residues of a bound peptide substrate. We have now determined two additional HNC complexes formed with the thiol inhibitor HSCH2CH(CH2Ph)CO-L-Ala-Gly-NH2 and another hydroxamate inhibitor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2, which were both refined to R-values of 0.183/0.198 at 0.240/0.225-nm resolution. The inhibitor thiol and hydroxamate groups ligand the catalytic zinc, giving rise to a slightly distorted tetrahedral and trigonal-bipyramidal coordination sphere, respectively. The thiol inhibitor diastereomer with S-configuration at the P1' residue (corresponding to an L-amino acid analog) binds to HNC. Its peptidyl moiety mimics binding of primed (P1'-P3') residues of the substrate. In combination with our first structure a continuous hexapeptide corresponding to a peptide substrate productively bound to HNC was constructed and energy-minimized. Proteolytic cleavage of this Michaelis complex is probably general base-catalyzed as proposed for thermolysin, i.e. a glutamate assists nucleophilic attack of a water molecule. Although there are many structural and mechanistic similarities to thermolysin, substrate binding to MMPs differs due to the interactions beyond S1'-P1'. While thermolysin binds substrates with a kink at P1', substrates are bound in an extended conformation in the collagenases. This property explains the tolerance of thermolysin for D-amino acid residues at the P1' position, in contrast to the collagenases. The third inhibitor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2, unexpectedly binds in a different manner than anticipated from its design and binding mode in thermolysin. Its hydroxamate group obviously interacts with the catalytic zinc in a favourable bidentate manner, but in contrast its isobutyl (iBu) side chain remains outside of the S1' pocket, presumably due to severe constraints imposed by the adjacent planar hydroxamate group. Instead, the C-terminal Ala-Gly-NH2 tail adopts a bent conformation and inserts into this S1' pocket, presumably in a non-optimized manner. Both the isobutyl side chain and the C-terminal peptide tail could be replaced by other, better fitting groups.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:X-ray structures of human neutrophil collagenase complexed with peptide hydroxamate and peptide thiol inhibitors. Implications for substrate binding and rational drug design. 773 83

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.
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PMID:Hydroxamic acids as potent inhibitors of endothelin-converting enzyme from human bronchiolar smooth muscle. 778 43

Zinc endopeptidase thermolysin can be inhibited by a series of phosphorus-containing peptide analogues, Cbz-Gly-psi (PO2)-X-Leu-Y-R (ZGp(X)L(y)R), where X = NH, O, or CH2; Y = NH or O; R = Leu, Ala, Gly, Phe, H, or CH3. The affinity correlation as well as an X-ray crystallography study suggest that these inhibitors bind to thermolysin in an identical mode. In this work, we calculate the electrostatic binding free energies for a series of 13 phosphorus-containing inhibitors with modifications at X, Y, and R moieties using finite difference solution to the Poisson-Boltzmann equation. A method has been developed to include the solvation entropy changes due to binding different ligands to a macromolecule. We demonstrate that the electrostatic energy and empirically derived solvation entropy can account for most of the binding energy differences in this series. By analyzing the binding contribution from individual residues, we show that the energy of a hydrogen bond is not confined to the donor and acceptor. In particular, the positive charges on Zn and Arg 203, which are not the acceptors, contribute significantly to the hydrogen bonds between two amides of ZGpLL and the thermolysin.
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PMID:Binding of phosphorus-containing inhibitors to thermolysin studied by the Poisson-Boltzmann method. 779 20

Specific proteolysis by the tetanus toxin light chain of a vesicle-associated membrane protein (VAMP) involved in exocytosis is thought to underlie its intracellular blockade of neurotransmitter release. To substantiate this mechanism, recombinant light chain was expressed as a maltose binding protein-light chain fusion product in Escherichia coli. After purification of affinity chromatography and cleavage with factor Xa, the resultant light chain was isolated and its identity confirmed by Western blotting and N-terminal sequencing. It exhibited activity similar to that of the native light chain in proteolyzing its target in isolated bovine small synaptic vesicles and in hydrolyzing a 62-residue synthetic polypeptide spanning the cleavage site of the substrate. The importance of Glu234 in the catalytic activity of the light chain, possibly analogous to Glu143 of thermolysin, was examined using site-directed mutagenesis. Changing Glu234 to Ala abolished the protease activity of the light chain, but its ability to bind the polypeptide substrate was retained. Each recombinant light chain could be reconstituted with the heavy chain of tetanus toxin, yielding the same level of disulfide-linked species as the two native chains. Whereas the toxin formed with wild-type light chain exhibited appreciable neuromuscular paralysis activity and mouse lethality, the equivalent dichain material containing the Ala234 mutant lacked neurotoxicity in both the in vitro and in vivo assays. Thus, these results demonstrate directly, for the first time, that the lethality of tetanus toxin and its inhibition of exocytosis in intact neurons are attributable largely, if not exclusively, to endoprotease activity.
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PMID:A single mutation in the recombinant light chain of tetanus toxin abolishes its proteolytic activity and removes the toxicity seen after reconstitution with native heavy chain. 791 29

The reported cDNA structure of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olson et al. Proc. Natl. Acad. Sci USA, 87:2284-2288, 1990). The calculated M(r) is 107,534 whereas the estimate by SDS-PAGE is approximately 130,000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors in the cDNA sequence for non-muscle MLCK and that the NH2-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH2-terminally blocked. A CNBr peptide derived from smMLCK contains the NH2-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating that Met-1 is present. Using a limited thermolysin digest we isolated an NH2-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH2-terminal Met being blocked by acetylation. The results demonstrate that the NH2-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating Met is not removed, but modified by alpha-NH2 acetylation of the translation product.
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PMID:Chicken smooth muscle myosin light chain kinase is acetylated on its NH2-terminal methionine. 793 65

The protective antigen (PA) component of anthrax toxin contains two sites that are uniquely sensitive to proteolytic cleavage. Cleavage at the sequence RKKR167 by the cellular protease furin is absolutely required for toxicity, whereas cleavage by chymotrypsin or thermolysin at the sequence FFD315 inactivates the protein, apparently by blocking the ability of PA to translocate the catalytic moieties of the toxins, lethal factor (LF) and edema factor (EF), to the cytosol of eukaryotic cells. To specify the role of the chymotrypsin-sensitive site of PA in the translocation of LF, we altered residues 313-315. None of the mutations in this region interfered with the ability of PA to bind to its cellular receptor, be cleaved by cell surface furin, and bind LF. Substitution of Ala for Asp315 or for both Phe313 and Phe314 reduced the ability of PA to intoxicate cells in the presence of LF by 3- and 7-fold, respectively. Substitution of Phe313 by Cys greatly reduced the rate of LF translocation and delayed toxicity. The rate at which the Cys-substituted PA killed cells was increased significantly by blocking the sulfhydryl group with iodoacetamide, suggesting that this added Cys interacts with cellular proteins and slows translocation of LF. Deletion of the 2 Phe rendered PA completely non-toxic. This deleted PA protein lacked the ability shown by native PA to form oligomers on cells and in solution and to induce release of 86Rb from Chinese hamster ovary cells. These results suggest that the chymotrypsin-sensitive site in PA is required for membrane channel formation and translocation of LF into the cytosol. PA double mutants were constructed that cannot be cleaved at either the furin or chymotrypsin sites. These PA proteins were more stable in Bacillus anthracis culture supernatants and may therefore be useful as a replacement for PA in anthrax vaccines.
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PMID:The chymotrypsin-sensitive site, FFD315, in anthrax toxin protective antigen is required for translocation of lethal factor. 796 69

The entomopathogenic fungus, Metarhizium anisopliae, produces three distinct types of proteinases during growth on cockroach cuticle. These were separated by analytical isoelectric focusing and characterized according to their substrate specificity and inhibition patterns as Pr1 subtilisin-like proteinases (four isoforms pI range approximately 9.3-10.2), a thermolysin-like metalloproteinase (pI approximately 7.3), and trypsin-like serine Pr2 proteinases (two major isoforms, pI approximately 4.4 and 4.9 and two minor isoforms, pI approximately 5.2). Preparative isoelectric focusing was used to separate the four Pr1(2) components produced during growth on cockroach cuticle with isoelectric points of 10.2 (m = 30.2 kDa), 9.8 (m = 28.5 kDa), 9.3 (m = 29.5 kDa), and 9.0 (m = 31.5 kDa). Two of the isoforms were also produced, at diminished levels, during growth on elastin or cellulose presumably as a result of carbon and nitrogen derepression. The pI 10.2 Pr1 differed from the other isoforms in preferring alanine over bulky hydrophobic groups at P2 and P3, in discriminating against proline at P2 and in its lack of sensitivity to tetra-butyl-oxycarbonyl-Gly-Leu-Phe-chloromethyl ketone. Differences in the N-terminal amino acid sequences confirmed that the four isoforms are related products of at least two distinct genes. The isoforms showed similar primary specificities, with the aromatic P1 phenylalanine being 10- to 16-fold more reactive than a P1 leucine residue reflected principally in Kcat. However, methionine (containing a long unsubstituted side chain) was also a good substrate for each isoform confirming the low selectivity of their S1 subsites. The isoforms all degraded a variety of solubilized cuticle proteins, with high-molecular-weight acidic proteins being preferentially hydrolyzed. The metalloproteinase is active against the Pr1 substrate succinyl-(Ala)2-Pro-Phe-7-amino-4-coumarin trifluoromethyl, but differs from the Pr1 isoforms in being inhibited by 1,10-phenanthroline and phosphoramidon. The potential role of the metalloproteinase in pathogenicity is discussed.
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PMID:Isoforms of the cuticle-degrading Pr1 proteinase and production of a metalloproteinase by Metarhizium anisopliae. 805 68


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